CN1589152A

CN1589152A - Novel human beta 2 integrin alpha subunit

Info

Publication number: CN1589152A
Application number: CNA018202500A
Authority: CN
Inventors: W·M·加拉丁; M·范德维伦
Original assignee: Icos Corp
Current assignee: Icos Corp
Priority date: 2000-10-13
Filing date: 2001-10-15
Publication date: 2005-03-02
Anticipated expiration: 2021-10-15
Also published as: KR20030044001A; AU2001296839B2; JP2004532800A; WO2002030980A2; WO2002030980A8; MXPA03003322A; CN100398152C; EP1325031A2; US6432404B1; NZ525402A; WO2002030980A3; AU9683901A; CA2425818A1

Abstract

The invention provides methods for reducing inflammation at the site of a central nervous system injury comprising the step of administering to an individual an effective amount of a small molecule that inhibits alpha-d binding. In particular, the methods of the invention comprising a central nervous system injury which is a spinal cord injury. Methods to treat spinal cord injury using alpha-d monoclonal antibodies are disclosed.

Description

Novel Human β 2 beta 2 integrin alpha subunits

The application is the unsettled U.S. Patent Application Serial of submitting on July 8th, 1,999 09/350,259 continuation part, 09/350,259 is unsettled U.S. Patent Application Serial 09/193 of submitting on November 16th, 1998,043 continuation part, 09/193,043 is the unsettled U.S. Patent Application Serial of submitting on October 3rd, 1,997 08/943,363 continuation part, 08/943,363 is continuation parts of the U.S. Patent Application Serial 08/605,672 of submission on February 22nd, 1996,08/605,672 authorize on October 6th, 1998 and to be United States Patent (USP) 5,817,515, it is the U.S. Patent Application Serial 08/362 of December in 1994 submission on the 21st, 652 continuation part, 08/362,652 authorizes on June 16th, 1998 and to be United States Patent (USP) 5,766,850, it is the continuation part of the U.S. Patent Application Serial 08/286,889 of submission on August 5th, 1994,08/286,889 authorize November 28 nineteen ninety-five and to be United States Patent (USP) 5,470,953, it is the U.S. Patent Application Serial 08/173 of December in 1993 submission on the 23rd, 497 continuation part, 08/173,497 authorizes August 1 nineteen ninety-five and to be United States Patent (USP) 5,437,958.Introduce these in full at this and apply for reference.

Background of invention

Integrin is the active film binding molecule that participates in cell adhesion of a class.Integrin be comprise with the non-covalent bonded α subunit of β subunit stride the film heterodimer.So far, at least 14 kinds of α subunits and 8 kinds of β subunits [summarizing in Springer Nature 346:425-434 (1990)] have been identified.The β subunit generally can combine with more than one α subunit, and the heterodimer with a common β subunit is categorized as the subtribe among the integrin group.

One class is limited to the human beta 2 integrin of expressing and is characterised in that common β in leukocyte ₂Subunit, as the result of this cell specific expression, so-called leukocyte integrin of these integrins or Leu-CAMs.Because these common β ₂Subunit, this class is called after β also ₂Integrin.β ₂The α subunit CD11a different with three kinds before the subunit (CD18), one of CD11b or CD11c separate together.The separation of the cDNA of coding people CD18 is described in Kishimoto, etal., Cell 48:681-690 (1987).In official's name of WHO, this heterodimer albumen is called CD11a/CD18, CD11b/CD18, and CD11c/CD18; In name commonly used, they are called as LFA-1 respectively, Mac-1 or Mol and p150,95 or LeuM5[Cobbold, etal., in Leukocyte Typing III, McMichael (ed), Oxford Press, p.788 (1987)].Reference β ₂The α subunit CD11a of integrin moves under CD11b and the CD11c reducing condition in electrophoresis, and apparent molecular weight is respectively 180kD, 155kD and 150kD, and cloned the DNA[CD11a of these subunits of encoding, Larson, et al., J.Cell Biol.108:703-712 (1989); CD 11b, Corbi, et al., J.Biol.Chem.263:12403-12411 (1988) and CD11c, Corbi, et al.EMBOJ 6:40234028 (1987)].At other species, comprise monkey and other primate [Letvin, etal., Blood 61:408-410 (1983)], mice [Sanchez-Madrid, et al., J.Exp.Med.154:1517 (1981)] and Canis familiaris L. [Moore, et al., Tissue Antigens36:211-220 (1990)] in identified people β ₂The supposition congener of beta 2 integrin alpha and β chain is to be determined by the approximate of molecular weight.

The absolute molecular weight of the supposition congener of verified other species is widely different [sees, Danilenko et al. for example, Tissue Antigens 40:13-21 (1992)], lacking under the situation of sequence information the definite dependency of there is no telling human beta 2 integrin subunit and the subunit in other species, identified.And, between different plant species, observed the variation of protein family number of members.For example in rabbit than in the people, separated more IgA isotype [Burnett, et al., EMBO be (1989) and Schneiderman J.8:4041-4047, et al., Proc.Natl.Acad.Sci. (USA) 86:75617565 (1989)].Equally, in the people, at least 6 kinds of variants [Karin and Richards, Nature299:797-802 (1982) and Varshney, the et al. of metallothionein have been identified, Mol.Cell.Biol.6:26-37, (1986)], and in mice, having only two kinds of such variants is tangible [Searle, et al., Mol.Cell.Viol.4:1221-1230 (1984)].Therefore, in species, exist a plurality of members of a protein family to be illustrated in and have corresponding family member in another species.

At β ₂In the concrete condition of integrin, in Canis familiaris L., observe the dog β of the people CD 18 of supposition ₂Homologue can form dimer [Danilenko, etal., supra] with 4 potential different α subunits.Cause monoclonal antibody by the antibody that produces with dog cell immunity of spleen mice, their immunoprecipitations temporarily be called people CD 18, CD 11a, the albumen of the dog congener of CD 11b and CD 11c, this is mainly based on similar but be not the molecular weight that is equal to.Another kind of anti-dog splenocyte antibody Ca11.8H2 identification and a kind of the 4th α sample dog subunit of immunoprecipitation, this subunit also can with β ₂The subunit combination is expressed but have unique molecular weight and be limited in a hypotype of differentiated tissues macrophage.

Cause two kinds of anti-alpha 2 integrin antibodies [Metlay, et al., J.Exp.Med.171:1753-1771 (1990)] by the antibody that produces with Mus dendritic cell immunity hamster.A kind of antibody 2E6 immunoprecipitation contains the advantage heterodimer of the minor band that subunit that approximate molecular weight is 180kD and 90kD and molecular weight ranges be 150-160kD.According to cell adhesion blocking-up research, suppose the Mus homologue of antibody 2E6 identification people CD18.The prompting mouse-anti is former to show the viewed different tissue distribution pattern with people CD11c/CD18 although the prompting of the antigenic molecular weight of N418 to the identification of the Mus congener of people CD11c/CD18, is further analyzed.

Can represent the dog of evaluation in the past or the Different Variation kind of Mus α subunit (as glycosylation or shearing variation) by the antigen of dog Ca11.8H2 antibody and Mus N418 antibody recognition.As selection, these antigens can be represented unique dog or Mus α subunit.When not having the specifying information of primary structure, these selections can not be distinguished.

In the mankind, CD11a/CD18 expresses on all leukocyte.CD11b/CD18 and CD11c/CD18 are limited to substantially on mononuclear cell, granulocyte, macrophage and NK cell (NK) cell and express, but CD11c/CD18 also detects on some B cell types.Generally, CD11a/CD18 preponderates on lymphocyte, and CD11b/CD18 preponderates on granulocyte, CD11b/CD18 preponderate on macrophage [referring to summary, Arnaout, Blood 75:1037-1050 (1990)].Yet for the activation and the differentiation state of each cell type, the expression of α chain is variable [referring to summary, Larson and Springer, Immunol.Rev.114:181-217 (1990)].

Employing can be blocked β ₂The adherent monoclonal antibody of integrin relevant cell is verified β ₂Integrin participant immunity and inflammatory reaction.For example, CD11a/CD18, CD11b/CD18 and D11c/CD18 be active to participate in the [Patarroyo that combines of NK cell (NK) cell and lymphoma and adenocarcinoma cell, et al., Immunol.Rev.114:67-108 (1990)], granulocyte is assembled [Nourshargh, et al., J.Immunol.142:3193-3198 (1989)], granulocyte dependent/non-dependent blood plasma seepage [Arfors, et al., Blood 69:338-340 (1987)], stimulated leukocytic chemotactic response [Arfors, et al., supra] and adhesion [Price, et al., the J.Immunol.139:4174-4177 (1987) and the Smith of leukocyte and blood vessel endothelium, et al., J.Clin.Invest.83:2008-2017 (1989)].β ₂The basic role of integrin in immunity and inflammatory reaction is being called in the clinical syndrome of leukocyte adhesion deficiency (LAD) clearly, and clinical manifestation wherein comprises recurrence and common life-threatening bacterial infection.LAD comes from β ₂The allos sudden change [Kishimoto, et al., Cell 50:193-202 (1987)] of subunit, the seriousness of this morbid state and β ₂The defect level of subunit expression is proportional.β ₂The formation of the complete integrin heterodimer of sudden change infringement.Interesting is, the specific antibody of verified at least a CD18 can form by vitro inhibition 1 type human immunodeficiency virus (HIV-1) syncytium, but this suppresses cutter system not clear [Hildreth and Orentas, Science 244:1075-1078 (1989)] really.This observed result is consistent with following discovery, i.e. the main counter receptor ICAM-1 of the CD11a/CD18 surface receptor [Greve, et al., Cell 56:839 (1989)] that also is the main family of rhinovirus serotype.

β ₂Integrin has been emphasized the necessity more fully understood of exploitation to such surface protein in conjunction with the importance of activity in people's immunity and inflammatory reaction.Identify the unknown member of this subfamily, and their counter receptor, and generation can change β ₂The bioactive polyclonal antibody of integrin or other solvable factor will be β ₂The treatment intervention of integrin immunity and inflammatory reaction provides practical methods.

Summary of the invention

In one aspect, the invention provides novel purification and isolating polynucleotide (for example DNA and rna transcription thing comprise sense strand and antisense strand), these polynucleotide encodings have α _dThe Novel Human β of inherent combination and/or immunological characteristic ₂Beta 2 integrin alpha subunit α _dAnd variant (promptly lack, add or replace analog).Preferred dna molecular of the present invention comprises the dna molecular of cDNA, genomic DNA and all or part of chemosynthesis.The previous preferred polynucleotide of order are the DNA that list among the SEQ ID NO:1, the polypeptide of its coding SEQ ID NO:2.The present invention also provides and comprises α _dThe recombiant plasmid of coded sequence and viral DNA construct (expression construct), wherein α _dThe coded sequence operability is connected with one or more homologies or allogenic transcription regulatory element.

The present invention people α that also provides and encode _dPolynucleotide show the separation of homology and the mice and the rat polynucleotide of purification.Preferred mice polynucleotide are listed in SEQ ID NO:52, and preferred rat polynucleotide are listed in SEQ ID NO:54.

Another aspect of the present invention is to provide with the energy express alpha _dThe DNA sequence conversion of the present invention of polypeptide or its variant or the protokaryon or the eukaryotic host cell of transfection.Host cell of the present invention is at a large amount of preparation α _dParticularly useful during polypeptide, α _dPolypeptide can separate from the culture medium of host cell itself or host cell growth.At its epicyte surface expression α _dThe host cell of polypeptide is preparing α as immunogen _dAlso particularly useful during special antibody.Preferably, use α _dThe host cell of transfection also carries out cotransfection and expresses a kind of β ₂Integrin makes heterodimer at surface expression.

The present invention also provides purification and isolating α _dPolypeptide and fragment thereof and variant.Preferred α _dPolypeptide is listed in SEQ ID NO:2.Novel α of the present invention _dProduct can separate from natural origin, but and α _dThe variant product is the same, preferably produces by recombination method with host cell of the present invention.Can select to be used for carrying out the host cell of regrouping process and/or to separate the α that post-treatment prepares complete glycosylation, part glycosylation and complete deglycosylation form by changing _dPolypeptide.α of the present invention _dPolypeptide variants can comprise water solublity and insoluble α _dPolypeptide comprises analog, and wherein one or more aminoacid are removed or replace: (1) α _dSpecial biologic activity or amynologic characteristic be forfeiture not, and preferably has one or more enhancings that have; (2) specific ligand/receptor in conjunction with or semiotic function by inactivation specifically.The present invention also provides the polypeptide of fusion, wherein α _dAminoacid sequence with from other amino acid sequence of polypeptide sequential expressions.The such fused polypeptide and the α of wild type _dCompare biology, biochemistry and/or immunological characteristic with modification.The present invention also comprises the polypeptide analog that comprises extra amino acid residue (for example lysine or cysteine) thereby be convenient to polymer formation.

The present invention also comprises specifically and α _dBonded polypeptide or non-peptide quasi-molecule.Preferred binding molecule comprises antibody (for example monoclonal and polyclonal antibody), counter receptor (for example film is relevant and soluble form) and other parts (for example natural existence or synthetic molecule), comprises that those are at α _dMonoclonal antibody and/or specificity counter receptor exist down competitively and α _dBonded molecule.Binding molecule is at purification α _dPolypeptide and evaluation express alpha _dCell type the time very useful.Binding molecule also can be used for regulating (promptly suppress, block or activate) α _dBody in conjunction with and/or signaling activity.

The present invention also provides and identifies α _dThe mensuration of binding molecule, comprise external test for example fixed ligands in conjunction with measure, solution is in conjunction with measuring and mensuration is got close in flicker; And based on for example double cross body Screening test of mensuration of cell, the Screening test of division (split) crossbred or the like.Provide phenotypic alternation as specificity binding interactions or the ruined result of specificity binding interactions in the host cell based on being determined at of cell, thereby indirectly quantitatively or measure some specificity binding interactions.

Evaluation can be regulated α _dThe external test of active antibody or other chemical compounds can comprise, for example, and immobilization α _dOr a kind of α _dBonded natural gametophyte (partner), the binding partner of on-fixedization is carried out detectable label, with binding partners incubation together, measure the influence of testing compound to labelling quantity, wherein under having under the situation of testing compound than the situation that does not have testing compound, bonded labelling reduces, and just illustrates that test agent is a kind of α _dBonded inhibitor.

The evaluation of another kind of type can be regulated α _dComprise with the external test of the chemical compound of ligand interaction, with α _dOr its fragment be immobilized onto bag by (or saturated) on a kind of solid support of fluorometric reagent, part is with the chemical compound labelling of a kind of energy fluorescence excitation, with immobilized α _dContact under the situation that has or do not exist a kind of adjusting chemical compound of inferring with the part of labelling, detect the light emission of fluorometric reagent, the light emission of fluorometric reagent is compared and can be influenced the photoemissive chemical compound of fluorometric reagent and just be accredited as the adjusting chemical compound when not regulating chemical compound and exist.In addition, also can be in mensuration with α _dPart immobilization and with α _dLabelling.

A kind of evaluation that the present invention comprises can be regulated α _dComprise with a kind of method of chemical compound of ligand interaction: transform or the transfection proper host cell with a DNA construct based on cell, contain a reporter gene under promoter control in this DNA construct, the transcription factor that this promoter is contained a DNA binding structural domain and an activation structure territory is regulated; In host cell, express coded portion or whole α _dFirst kind of hybrid dna sequence with first kind of fusion in the DNA binding structural domain of transcription factor or activation structure territory; The DNA binding structural domain of all or part of and transcription factor of expression coding part or second kind of hybrid dna sequence in activation structure territory in host cell, it does not mix first kind of fusion; Exist or do not infer under the situation of regulator by measuring, the output of reporter gene product in host cell is come detector ligand and α _dCombination in particular host cell, thus the adjusting chemical compound that evaluation is inferred is to α _dWith the influence of ligand interaction, and the compound identification that will change reporter gene output when not regulating chemical compound is for regulating chemical compound.What preferably use in mensuration here is lexA promoter, lexA DNA binding structural domain, GAL4 transactivation domain, lacZ reporter gene and a kind of yeast host cell.

A revision of said determination can be used for separating coding is a kind of can be in conjunction with α _dProteic polynucleotide, comprise: transform or the transfection proper host cell with a DNA construct, contain a reporter gene under promoter control in this DNA construct, the transcription factor that this promoter is contained a DNA binding structural domain and an activation structure territory is regulated; In host cell, express coded portion or whole α _dFirst kind of hybrid dna sequence with first kind of fusion in the DNA binding structural domain of transcription factor or activation structure territory; The DNA binding structural domain of all or part of and transcription factor of expression coding part or second kind of hybrid dna sequence in activation structure territory in host cell, it does not mix first kind of fusion; Detect α in particular host cell by the output that detects reporter gene product in the host cell _dConjugated protein and α _dCombination; And from particular host cell, separate coding for alpha _dProtein-bonded second kind of hybrid dna sequence.

Use in the preferred embodiment that divides the crossbred Screening test at one, the invention provides a kind of evaluation α _dAlbumen or its fragment and α _dThe method of the conjugated protein or bonded inhibitor of its fragment, this method comprises following steps: (a) transform or the described host cell of transfection with first DNA expression construct, this DNA expression construct comprises the suppressor gene of proteic first selectable marker gene of first selected marker of coding and a repressor protein of coding, wherein said suppressor gene is transcribed under the control a promoter, (b) transform or the described host cell of transfection with second DNA expression construct, this DNA expression construct comprises second proteic second selectable marker gene of selected marker of coding and the 3rd proteic the 3rd selectable marker gene of selected marker of coding, wherein said the 3rd marker gene transcribing under the control at an operon, described operon is subjected to described repressor protein effect specifically, described the 3rd the proteic expression of selected marker of reduction thereby described repressor protein and described promoter interact; (c) transform or the described host cell of transfection with the 3rd DNA expression construct, this DNA expression construct comprises the 4th proteic the 4th selectable marker gene of selected marker of coding and α of coding _dThe DNA binding structural domain of albumen or its fragment and a transcription activating protein or the trans-acting domain of described transcription activating protein are with the α of frame _dAntigen-4 fusion protein gene; (d) transform or the described host cell of transfection with the 4th DNA expression construct, this DNA expression construct comprises the 5th proteic the 5th selectable marker gene of selected marker of coding and α of coding _dThe DNA binding structural domain of conjugated protein or its binding fragment and described transcription activating protein or the trans-acting domain of described transcription activating protein are with second antigen-4 fusion protein gene of frame, and this DNA binding structural domain or trans-acting domain are not included in first antigen-4 fusion protein gene; (e) described host cell is being allowed described α _dAlbumen or its fragment and α _dCultivate under conjugated protein or the condition that its binding fragment is expressed, thereby make described α _dAlbumen or its fragment and α _dConjugated protein or its binding fragment interacts, and makes described DNA binding structural domain and described trans-acting domain approaching, reassembles into described transcription activating protein; Described transcription activating protein acts on described promoter, and the expression of described repressor protein is improved; Described repressor protein and described promoter interact, and the 3rd selected marker albumen is not expressed; (f) shortage of the described selection gene expression of detection; (g) there is a kind of described α in described host cell _dAlbumen or its fragment and described α _dCultivate under the condition of the conjugated protein or bonded testing inhibitor of its fragment; (h) relatively exist and do not exist under the situation of described testing inhibitor, the proteic expression of described selected marker, the proteic expression of wherein said selected marker reduces the explanation testing inhibitor can suppress described α _dAlbumen or its fragment and described α _dThe combination of conjugated protein or its binding fragment, thus described transcription activating protein can not be recombinated, and the expression of described repressor protein does not increase, and said operon increases the proteic expression of described selected marker.

The present invention includes different genes and regulate sequential coding at the host cell of unique DNA molecule and one or more suppressor gene, selectable marker gene, α _dAntigen-4 fusion protein gene and α _dBinding gene coded host cell in different DNA expression construct.In a preferred embodiment, host cell coding suppressor gene, selectable marker gene, α _dAntigen-4 fusion protein gene and α _dThe DNA of binding-protein gene transforms or transfection, and said gene all is coded on the different expression construct.No matter import what DNA expression construct, the DNA expression construct of each conversion or transfection further comprises a selectable marker gene sequence, and the expression of this selectable marker gene sequence is used for that proof transforms or transfection is finished really.The coding selection marker gene is different with tet operon transcriptional regulatory selected marker on each conversion or the transfection DNA expression construct, its difference is that being expressed in this preferred embodiment of selectable marker gene under the adjusting of tet operon is core, be that the regulation and control that the tet operon is expressed selectable marker gene provide the phenotypic alternation that can measure in host cell, in order to identify binding-protein inhibitors.Each transform or the transfection DNA expression construct on the coding selection marker gene provide and be used as that each DNA expression construct successfully transforms or the decision thing of transfection.Preferred host cell of the present invention comprises the saccharomyces cerevisiae (S.cerevisiae) of called after YI596 and YI584, these two kinds of bacterium are preserved in American type culture collection (ATCC) on August 13rd, 1996,12301 Parklawn Drive.Rockville.Maryland 20852, and be given preserving number ATCC 74384 and ATCC74385 respectively.

Host cell of the present invention comprises anyly can express as above required α _dAlbumen and α _dConjugated protein and the promoter that function is arranged that can be expressed by aforementioned adjusting heterologous protein and operon sequence transforms or the host cell of transfection.In a preferred embodiment, host cell can be the cell in mammal, insecticide or yeast source.At this, most preferred cell is a yeast cells.Preferred yeast cell of the present invention can be selected from the various bacterial strains that comprise the saccharomyces cerevisiae of introducing in the table 1.Other yeast specie comprises schizosaccharomyces pombe (S.pombe), newborn kluyveromyces (K.lactis), pichia pastoris phaff (P.pastoris), Ka Ersibai yeast (S.carlsbergensis) and white candida mycoderma (C.albicans).Preferred mammal class host cell of the present invention comprises the transfectional cell series in Chinese hamster ovary (CHO) cell, COS, HeLa, 3T3, CV1, LTK, 293T3, Rat1, PC12 or any other people or Rodents source.Preferred insect cell line comprises the SF9 cell.

In a preferred embodiment, selectable marker gene is regulated by an operon, an and enzyme in the required route of synthesis of the nutrition of the described host cell of encoding, like this, for lacking described said host cell of growing on must the culture medium of nutrition, the proteic expression of described selected marker needs.Like this, in a repressor protein and the interactional preferred embodiment of operon, transcribing of selectable marker gene reduced, and host cell can not be grown on the essential culture medium of nutrition but can grow on must the culture medium of nutrition and identify containing this lacking by it.In a most preferred embodiment, selectable marker gene coding HIS3 albumen transforms or the host cell of transfection is selected after growing on the culture medium of histidine existing or do not contain with the DNA expression construct of coding HIS3.Yet the present invention can comprise the selectable marker gene that any other is regulated by an operon.Other genes comprise, for example, and the gene of needed plurality of enzymes in the various approach of the essential nutrition that URA3, LEU2, LYS2 or those any generations of encoding can fully be removed from growth medium.In addition, traditional reporter gene for example chloramphenicol acetyltransferase (CAT), Lampyridea luciferase, beta galactosidase (β-gal), secreting type alkali phosphatase (SEAP), green fluorescent protein (GFP), human growth hormone (hGH), β-glucuronic acid enzyme, neomycin, hygromycin, thymidine kinase (TK) etc. also can be used for the present invention.

In a preferred embodiment, host cell comprises the repressor gene of a coding tetracycline resistance protein, and this albumen acts on the tet operon, and the expression of selectable marker gene is reduced.But the present invention also comprises other repressors and operon beyond tet repressor and the operon, for example escherichia coli trp repressor and operon, his repressor and operon and lac repressor and operon.

The DNA binding structural domain of fusion rotein can be from same transcription factor or from different transcription factor, as long as at α with the transactivation domain component _dAnd α _dConjugated protein in conjunction with the time make these two kinds of domains near forming the transcription activating protein that function is arranged, this albumen improves the expression of repressor protein expeditiously.A high efficiency transcription activating protein is defined as containing simultaneously a bonded DNA binding structural domain of the promoter sequence high-affinity that can show and be identified and a transactivation domain that the required record changer producing protein of expression suppressor gene mRNA is had high-affinity.The DNA binding structural domain component of fusion rotein of the present invention can be from any different proteins, for example, and LexA or Ga14.Similarly, the trans-activation component of fusion rotein of the present invention can comprise as Ga14 or VP16 from multiple different transcription activating protein.

The promoter sequence that adjusting repressor protein of the present invention is transcribed can be anyly can drive the sequence of transcribing in selected host cell.Promoter can be specifically by the DNA sequence of selected DNA binding structural domain identification of the present invention, or any other can with the DNA binding structural domain of fusion rotein with the interactional DNA sequence of high-affinity.In a preferred embodiment of the invention, promoter sequence of the present invention is a HIS3 or an alcoholdehydrogenase (ADH) promoter.Here in the most preferred embodiment, the present invention has used an ADH promoter.But the present invention includes other a large amount of promoteres, for example comprise, come since coding HIS3, ADH, the isogenic promoter sequence of URA3, LEU2.

Method of the present invention comprises the variation among any all above-mentioned host.Especially, the present invention includes following method: wherein host cell is a kind of yeast cells; Selectable marker gene coding HIS3; Selectable marker gene transcribe the adjusting that is subjected to the tet operon; Repressor gene coding tetracycline resistance protein; Tetracycline resistance protein transcribe the adjusting that is subjected to the HIS3 promoter; The DNA binding structural domain is from LexA; Transactivation domain is from VP16.In another embodiment, the present invention includes following method: host cell is a kind of yeast cells; Selectable marker gene coding HIS3; Selectable marker gene transcribe the adjusting that is subjected to the tet operon; Repressor gene coding tetracycline resistance protein; Tetracycline resistance protein transcribe the adjusting that is subjected to the alcoholdehydrogenase promoter; The DNA binding structural domain is from LexA; Transactivation domain is from VP16.

In other embodiments of the present invention, host cell is a kind of mammalian cell, and its change comprises, uses the mammalian DNA expression construct to come coding for alpha _dAnd α _dIn conjunction with antigen-4 fusion protein gene, suppressor gene and selectable marker gene, and the selectable marker gene (that is, neomycin, hygromycin, thymidine kinase) of use coding antibiotic or drug resistance labelling.

Have at least three kinds of dissimilar libraries to be used for identifying micromolecular regulator.They comprise: (1) chemical library, (2) natural product libraries, the combinatorial library that (3) are made up of peptide, oligonucleotide or organic molecule at random.

The chemistry library is formed by known compound or by the analog that natural product screening is accredited as the chemical compound of " target ".Natural product libraries is the gleanings of microorganism, animal, plant or aquatic organism, above-mentioned organism is used for preparing as follows the mixture that is used for screening: (I) from soil, plant or aquatic microorganisms fermentation and extracting culture fluid, (II) extracting plant or aquatic organism.Combinatorial library is made up of the mixture of a large amount of peptides, oligonucleotide or organic compound.They can relatively easily be prepared with traditional automatic synthesis method, PCR, clone or suitable synthetic method.Wherein the most interesting is peptide and oligonucleotide combinatorial libraries.Other interesting libraries comprise peptide, albumen, peptide mimics, how parallel synthetic gleanings, reorganization and polypeptide libraries.

The present invention also comprises production α _dThe hybridoma cell line of specific antibody.Producing the technology of the hybridoma of energy secrete monoclonal antibody knows in this area.Hybridoma cell line can be at the α with purification _d, α _dVariant or can be at its epicyte surface expression α _dOr be prepared behind a kind of animal of the cellular immunization of its variant.The immunogen cell type is included in expression in vivo α _dCell or express alpha in vivo usually _dThe prokaryotic cell or the eukaryotic cell lines of transfection.Here the preferred antibody of the present invention is by called after 169A, 169B, 170D, 170F, 170E, 170X, 170H, 188A, 188B, 188C, 188E, 188F, 188G, 188I, 188J, 188K, 188L, 188M, 188N, 188P, 188R, 188T, 195A, 195C, 195D, 195E, 195H, 197A-1,197A-2,197A-3,197A-4,199A, 199H, 199M, 205A, 205C, 205E, 212A, 212D, 217G, 217H, 217I, 217K, 217L, 217M, 226A, 226B, 226C, 226D, 226E, 226F, 226G, 226H, 226I, 236A, 236B, 236C, 236F, 236G, 236H, 236I, 236K, 237L, 237L, 236M, 240F, the hybridoma secretion of 240G and 240H.

Pass through α _dDNA and aminoacid sequence is open and the value information that provides also is specified.In a series of examples, disclosed α _dThe cDNA sequence makes separation of human α _dGenome sequence comprises that the transcriptional control element of genome sequence becomes possibility.The present invention also comprises α _dThe evaluation of the DNA of allele variant and allos species (for example rat and mice).People α _dSeparating of genomic DNA and allos species DNA can under the condition of appropriateness strictness, be used all or part of α with the DNA/DNA hybridization technique of standard _dThe cDNA sequence is screened a suitable library as probe and is finished.In addition, use oligonucleotide primers based on known cDNA sequential design to carry out polymerase chain reaction (PCR) and also can be used to amplification and identified gene group α _dDNA sequence.Coding for alpha _dPolypeptide comprises that the synthetic DNA of its fragment and other variants can be produced with traditional synthetic method.

DNA sequence information of the present invention also can be used to produce the α that can not express function by homologous recombination or " knocking out " strategy [be seen in as, Kapecchi, Science 244:1288-1292 (1989)] _dPolypeptide or the α that expresses a kind of variation _dThe rodent of polypeptide.Such rodent can be used as research α in the body _dAnd α _dThe model of regulator.

DNA of the present invention and aminoacid sequence also make analyzes α _dIn participate in bonded epi-position of counter receptor and adjusting actively rather than participate in bonded epi-position actively becoming possibility.The evaluation that participates in the epi-position of film signal conduction is also contained in the scope of the present invention.

DNA of the present invention also can be used to detect express alpha _dThe cell type of polypeptide.Use α _dDNA detection α _dThe standard DNA of RNA/RNA hybridization technique can be used to determine α in a kind of cell _dThe composition level of transcribing and respond to inside or the transcriptional level of outside agent changes.And follow, can modified alpha _dThe evaluation of the reagent of transcribing or translating can be used for assessing its potential treatment or preventive values again.DNA of the present invention can also use α _dDNA pair cell RNA carries out in situ hybridization and determines α in the cell mass of complexity and tissue _dThe celluar localization of specificity information.

DNA of the present invention also can be used to identify and shows and people α _dThe inhuman polynucleotide sequence of sequence homology.Obtained inhuman α _dDNA sequence just can be set up the animal model (comprise, for example, transgenic models) of robot system.

As another aspect of the present invention, α _dMonoclonal antibody specific and polyclonal antibody can be used for α _dBe positioned the immunohistochemical analysis of each cell in subcellular fraction chamber or the tissue.This para-immunity group fractional analysis is being located α with the in situ hybridization coupling _dThe α of gene _dParticularly useful when mRNA and polypeptide product.

Identifying can express alpha _dCell type exploitation therapeutic and preventative reagent are had the effect of deriving significantly.Expect α of the present invention _dAssociated products can be used for treating that macrophage is the disease of Fundamentals in its lysis.In the art, the animal model of many pathological states relevant with macrophage activity has obtained introducing.For example, Jutila, et al., J.LeukocyteBiol.54:30-39 (1993) have reported in mice, raise macrophage in chronic and acutely inflamed site.Adams, et al., [Transplantation 53:1115-1119 (1992) and Transplantation 56:794-799 (1993)] described in rat, the arteriosclerotic model of transplanting after the allograft of anisotropy abdominal part heart.Rosenfeld, et al., [Arteriosclerosis 7:9-23 (1987) and Arteriosclerosis 7:24-34 (1987)] have introduced with a kind of food that adds cholesterol and have fed, and induce atherosclerosis in rabbit.Hanenberg, et al., [Diabetologia 32:126-134 (1989)] have reported the spontaneous development of insulin dependent diabetes mellitus (IDDM) in the BioBreeding rat.Yamada et al., [Gastroenterology 104:759-771 (1993)] have been introduced in rat and have been induced inflammatory bowel, chronic granuloma behind injection streptococcus Peptidoglycan-polysaccharide polymer.Cromartie, etal., [J.Exp.Med.146:1585-1602 (1977)] and Schwab, et al., [Infection and Immunity 59:4436-4442 (1991)] have reported and injected the arthritis that the streptococcus cell wall protein causes being characterized as periphery arthritis and follow-up destruction of joint in rats.At last, Huitinga, et al., [Eur.J.Immunol 23:709-715 (1993) has introduced the experiment type allergic encephalomyelitis of Lewis rat, and this is a kind of model of multiple sclerosis.In above-mentioned every kind of model, α _dAntibody, other α _dConjugated protein or α _dSoluble form be used to alleviate morbid state, supposition is by the macrophage deactivation.

The invention provides the pharmaceutical composition of these diseases of treatment and other diseases state.The purpose of design of pharmaceutical composition is to suppress α _dAnd the interaction between its part comprises α _dVarious solubilities and film combining form (comprise and participate in α actively _dBonded complete α _dPolypeptide or its fragment), the α of solubility and film combining form _dConjugated protein (comprising antibody, part etc.), α _dIn conjunction with in the active cell or extracellular regulator and/or α _dAnd/or α _dThe regulator that ligand polypeptide is expressed comprises the regulator of transcribing, translate, translate transportation in post-treatment and/or the cell.

The present invention comprises that also treatment relates to α _dIn conjunction with or express alpha _dThe method of the disease gathered of cell part, in the method, provide the pharmaceutical composition of the present invention of some to the patient who suffers from said disease, its quantity is enough regulated α _dIn conjunction with level or adjusting express alpha _dThe gathering of cell type.Therapeutic Method of the present invention is applied to following morbid state, includes but not limited to type i diabetes, atherosclerosis, multiple sclerosis, asthma, psoriasis, pneumonia, adult respiratory distress syndrome and rheumatoid arthritis.

The present invention also provides the method for the macrophages infiltration that suppresses the central nervous system injury site, comprises the anti-α that gives individual effective dose _dThe step of monoclonal antibody.On the one hand, this method comprises use blocking-up α _dAnd the bonded anti-α between the binding partners _dMonoclonal antibody.In one embodiment, binding partners is VCAM-1.In a preferred embodiment, anti-α _dMonoclonal antibody is selected from by the excretory monoclonal antibody of hybridoma 226H with by the excretory monoclonal antibody of hybridoma 236L.In a most preferred embodiment, method of the present invention is used for central nervous system injury, and this damage is spinal cord injury.

The present invention further provides the method that reduces the inflammation in central nervous system injury site, comprised the anti-α that gives individual effective dose _dThe step of monoclonal antibody.On the one hand, this method comprises use blocking-up α _dAnd the bonded anti-α between the binding partners _dMonoclonal antibody.In one embodiment, binding partners is VCAM-1.In a preferred embodiment, anti-α _dMonoclonal antibody is selected from by the excretory monoclonal antibody of hybridoma 226H with by the excretory monoclonal antibody of hybridoma 236L.In a most preferred embodiment, method of the present invention is used for central nervous system injury, and this damage is spinal cord injury.

Hybridoma 226H and 236L are by American type culture collection, and 10801 UniversityBoulevard, Masassas, VA 20110-2209 receive on November 11st, 1998 under budapest treaty, and preserving number is respectively HB12592 and HB-12593.

The present invention also provides the method for regulating macrophage or spleen phagocyte release TNF α, comprises the immunologic opsonin α that makes described phagocyte contact effective dose _dThe step of monoclonal antibody.One preferred aspect, method of the present invention comprises uses the anti-α of immunologic opsonin _dMonoclonal antibody, described anti-α _dMonoclonal antibody is selected from by the excretory monoclonal antibody of hybridoma 205C with by the excretory monoclonal antibody of hybridoma 205E.

In the method for being considered of the present invention, useful antibody comprises anti-α _dThe fragment of monoclonal antibody comprises for example Fab or F (ab ') ₂Fragment.The present invention also comprises the method for utilizing modified antibodies.The antibody of modifying comprises, for example, the antibody of single-chain antibody, chimeric antibody and CDR grafting comprises the chemical compound of the CDR sequence that contains specific recognition polypeptide of the present invention and humanized antibody.Also considered to comprise the method for end user's antibody.The technology of evaluation and isolating human antibodies is open hereinafter.

The present invention also provides the method for the macrophages infiltration that suppresses the central nervous system injury site, comprises the inhibition α that gives individual effective dose _dBonded micromolecular step.Particularly, method of the present invention relates to central nervous system injury, and this damage is spinal cord injury.From foregoing library, identify and be specific to α with having separated _dBonded micromolecule.

The present invention further provides the method for the inflammation that reduces the central nervous system injury site, comprise the inhibition α that gives individual effective dose _dBonded micromolecular step.Particularly, method of the present invention relates to central nervous system injury, and this damage is spinal cord injury.From foregoing library, identify and be specific to α with having separated _dBonded micromolecule.

The present invention further comprises the method that detects and diagnose clone disease, may further comprise the steps: obtain tissue sample from the patient; Use anti-α _dMonoclonal antibody dyes to sample, and the pattern that will dye compares with the dyeing pattern that obtains tissue from known normal donor.In the time can detecting two kinds of dyeing differences between the tissue sample, can further check the patient whether may suffer from clone disease.

The present invention also considers α _dAs removing at cell surface expression α _dThe target of population of pathogenic cells.On the one hand, clone and expressed α in the scope of people's isotype of complement fixation _dThe hypervariable region of monoclonal antibody.In this way clone the hypervariable region and will be α _dBinding partners is provided, in it carries out body in conjunction with the time, will cause complement combination and cell death subsequently.Perhaps, will resist α _dMonoclonal antibody and cytotoxic compound coupling, the α on antibody and the pathogenic cell type _dCombination will cause cell death.

The present invention also provides the anti-α by the victim's effective dose that gives spinal cord injury _dMonoclonal antibody and promote the exercise recovery after the spinal cord injury or suppress the method for motion infringement or constrained motion obstacle.Also relate to anti-α by the victim's effective dose that gives spinal cord injury _dMonoclonal antibody and the autonomic nerve after limiting spinal cord injury and the method for sensory disturbance.In one embodiment, anti-α _dMonoclonal antibody is 217L or 226H antibody.In another embodiment, anti-α _dMonoclonal antibody combines α with 217L or 226H competition _dIn another embodiment, anti-α _dMonoclonal antibody suppresses α _dWith α _dThe part combination.The anti-α that relates to _dMonoclonal antibody comprises ICAM-R and VCAM-1.Exemplary spinal cord injury by these method treatments comprises the damage that relates to the spinal cord compression.

The accompanying drawing summary

A large amount of other aspects of the present invention and advantage will show with reference to the accompanying drawings with following description, in the accompanying drawings:

Figure 1A to 1D comprises human amino acid sequence C D11b (SEQ ID NO:3), CD11c (SEQID NO:4) and α _dThe comparison of (SEQ ID NO:2).

Detailed Description Of The Invention

The present invention with following with from a human spleen cell cDNA library, separate coding for alpha _dThe relevant embodiment of cDNA illustrate.More particularly, embodiment 1 has illustrated and has used anti-dog α _TM1Antibody manages to detect a kind of homologous people's albumen.Embodiment 2 describes purification dog α in detail _TM1Check order with the N-terminal of polypeptide and to be designed for pcr amplification dog α _TM1The oligonucleotide primers of gene.Embodiment 3 has spoken of large scale purification and has been used for the inner dog α that checks order _TM1, be used for designing other PCR primer.Embodiment 4 has introduced use PCR and the inner primer dog α that increases _TM1A fragment of gene.Embodiment 5 has spoken of coding people α _dThe clone of cDNA sequence.Embodiment 6 has introduced express alpha _dThe people's tissue of mRNA and the Northern blot hybridization of cell.Embodiment 7 describes people α in detail _dThe structure of expression plasmid and with the plasmid transfection COS cell that obtains.Embodiment 8 has spoken of α in the rotaring redyeing COS cell _dThe elisa assay of expressing.Embodiment 9 has introduced personnel selection α _dThe facs analysis of the COS cell of expression plasmid transfection.Embodiment 10 has spoken of α in the COS cell of CD18 and cotransfection _dBonded immunoprecipitation.Embodiment 11 relates to α _dThe stable transfection of expression structure in Chinese hamster ovary cell.Embodiment 12 has spoken of α _dCD18 rely on the combining of ICAIU ICAM-R, a kind of ICAM-R mutain and complement factor iC3b.Embodiment 13 has introduced evaluation α _dScreening test is got close in the flicker of the inhibitor of part/anti-ligand interaction or reinforcing agent (being regulator).Embodiment 14 has spoken of the structure coding for alpha _dThe structure of the expression plasmid of soluble form and the binding analysis of expression product.Embodiment 15 relates to α _dSpecific polyclonal serum and MONOCLONAL ANTIBODIES SPECIFIC FOR.Embodiment 16 has introduced use α _dThe flow cytometry of monoclonal antibody.Embodiment 17 has spoken of α _dExpression on the person monocytic cell.Embodiment 18 has introduced and has used anti-α _dPolyclonal serum is analyzed α _dTissue distribution, α _dIn the expression on the peripheral blood lymphocyte, expression in inflammation and non-inflammation synovial membrane, from the expression among breast carcinoma patient's ill lung regulating liver-QI, people's bone marrow and the PBMC.Embodiment 19 has spoken of α _dIn vivo with the rise of vivoexpression.Embodiment 20 has introduced demonstration and people α _dThe separation of the homologous rat cdna sequence of gene order.Embodiment 21 has spoken of rat α _dThe tissue specific expression of mRNA.Embodiment 22 relates to the rat α of total length _dExpression plasmid, rat α _dI domain expression plasmid comprises the structure of I domain/IgG fusion rotein, at the MONOCLONAL ANTIBODIES SPECIFIC FOR of total length and I domain fusion rotein with at the rat α that merges with human IgG 4 _dThe preparation of the polyclonal antiserum of I domain sequence.Embodiment 23 has introduced the specificity of monoclonal antibody 199M.Embodiment 24 has shown use expression rat α _dThe result of T cell proliferation test of macrophage.Embodiment 25 has introduced myeloid rat α _dImmunoprecipitation.Embodiment 26 has introduced rat α _dExpression in several animal models.Embodiment 27 relates to a kind of use α _dMonoclonal antibody suppresses the cracked test of target cell of NK-tumor cell induction.Embodiment 28 has spoken of demonstration and people α _dThe separation of the homologous mice cDNA of gene order sequence.Embodiment 29 has introduced the mice α that other being used to proves sequence analysis _dCDNA clone's separation.Embodiment 30 relates to the in situ hybridization analysis of multiple mouse tissue, determines the people α that infers _dThe tissue of mice congener and cell specific expression.Embodiment 31 introduces the people α that coding is inferred _dThe preparation of the expression construct of mice congener.Embodiment 32 has spoken of the design of " knocking out " mice, wherein the people α that encodes and infer _dThe gene of mice congener is destroyed.Embodiment 33 has introduced demonstration and people α _dHomologous rabbit cDNA clone's separation.Embodiment 35 relates to the antigenic sign of monoclonal antibody 217L identification.Embodiment 36 has introduced the animal model of human disease's state, has wherein analyzed α _dThe treatment ability of regulating.Embodiment 37 has introduced α _dExpression in the animal model morbid state.Embodiment 38 speaks of α _dEffect in spinal cord injury.Embodiment 39 has described α _dExpression in clone disease.Embodiment 40 relates to from express alpha _dRat spleen cells discharge TNF α.Embodiment 41 has described and has used α _dMonoclonal antibody is regulated the release of TNF α from splenocyte.Embodiment 42 has identified α _dExpression on the eosinophilic granulocyte.Embodiment 43 relates to further evaluation α _dWith combining of VCAM-1.Embodiment 44 has described α _dAs the target of removing population of pathogenic cells.

Embodiment 1

Attempt detecting dog α _TM1People's congener

Detection is at dog α _TM1Monoclonal antibody Ca11.8H2 (Moore waits above) to the cross reaction of human peripheral lymphocyte, manage to identify a kind of dog α _TM1People's congener.Cellular preparations (is generally 1 * 10 ⁶Cell) with the control antibodies (10 μ g/ml) of the Mus IgG feminine gender of undiluted hybridoma supernatant or a kind of purification at incubation under the condition that has 0.1% Hydrazoic acid,sodium salt on ice.(CA) incubation detects bonded monoclonal anti body and function for Vector Laboratories, Burlingame with the anti-Mus IgG of the link coupled horse of the FITC of 6 μ g/ml subsequently.Painted cell is fixed with the 2%w/v paraformaldehyde in the phosphate-buffered saline (PBS), and (Becton Dickinson, Mountain View CA) analyzes with Facstar Plus fluorescence-activated cell sorter.Usually, amplify 10,000 cells of analysis with logarithm and measure fluorescence intensity.

The result shows, Ca11.8H2 not with the albumen cross reaction of human peripheral lymphocyte surface expression, and the superfluous natural disposition dog peripheral blood lymphocyte of control cells all is dog α basically _TM1Positive.

Since at the monoclonal antibody Ca11.8H2 of dog α subunit not with the cross reaction of people's congener, separate dog α _TM1DNA it is believed that it is necessary for the people's gene that separates the correspondence that may exist.

Embodiment 2

Affinity purification dog α _TM1Be used for the N-terminal order-checking

Affinity purification dog α _TM1Measure the N-terminal aminoacid sequence, be used for oligonucleotide probe/design of primers.In brief, will resist α _TM1Monoclonal antibody Ca11.8H2 is coupled to that (CA), albumen separates by specific protein-protein interaction for BioRad, Hercules on the Affigel_10 chromatography resin.Program according to BioRad recommends is coupled to the concentration of antibody by the every ml resin of about 5mg antibody on the resin.After the coupling reaction, remove unnecessary antibody, resin seals with the ethanolamine of the long-pending 0.1M of triploid.Use phosphate-buffered saline (PBS) washing resin of thirtyfold volume then.

Canis familiaris L. spleens of 25 grams at 250ml 25mM Tris-HCl, pH8.0, contain 0.32M sucrose and contain homogenate in the buffer of protease inhibitor.Made nuclear and cell residue precipitation in centrifugal 15 minutes at 1000g.100,000g precipitated film in centrifugal 30 minutes from supernatant.Film is deposited in the 200ml lysis buffer (50mM NaCl, 50mM borate, pH8.0,2%NP-40) again suspends, and incubation on ice 1 hour.100,000g precipitated insoluble matter in centrifugal 60 minutes then.10 milliliters of limpid supernatant are changed in the polypropylene test tube of a 15ml who contains the link coupled Affigel_10 resin of the above-mentioned Ca11.8H2 of 0.5ml.Then test tube is incubated overnight 4 ℃ of rotations, follows D-PBS washing resin with 50 times of volumes.Then resin is changed in the microcentrifugal tube, in 1ml contains Laemmli (irreducibility) sample buffer of 0.1M Tris-HCl, pH6.8,2%SDS, 20% glycerol and 0.002% bromophenol blue, boiled 10 minutes.The centrifugation resin also discards, and (Sigma, St.Louis MO) handle supernatant, and last 7% polyacrylamide gel with the beta-mercaptoethanol of 1/15 volume.Isolating albumen transfer to as follows the Immobilon pvdf membrane (Millipore, Bedford, MA) on.

Gel washing in deionized, the filtering water of Millipore_ is once containing the 10mM 3-[hexamethylene amino of 10% methanol]-1-propane sulfonic acid (CAPS), pH10.5 transfering buffering liquid in balance 15-45 minute.The Immobilon film soaks with methanol, and uses the filtered water rinsing, in the CAPS transfering buffering liquid balance 15-30 minute then.Preliminary transfer was carried out 3 hours at 70 volts with the BioRad transfer device.Remove the Immobilon film after the transfer, and in filtering 0.1%R250 coomassie dyestuff, dyeed 10 minutes.Film decolours three times in 50% methanol/10% acetic acid, each 10 minutes.After the decolouring, film washs in filtered water, and air drying.

Detected the protein band of about 150kD, 95kD, 50kD and 30kD.Infer that 50kD and 30kD band are that antibody pollutes generation.Then 150kD and 95kD band is carried out the N-terminal order-checking, but 95kD albumen seals, hindered order-checking.Downcut the 150kD protein band from film, (Foster City, CA) Model 473A protein sequencing instrument checks order according to the indication of manufacturer directly to use Applied Biosystems.The aminoacid sequence that is obtained adopts the name of single-letter aminoacid to list in SEQ ID NO:5.

FNLDVEEPMVFQ (the SEQ ID NO:5) sequence of being identified comprises the FNLD sequence signature [Tamura, etal., J.Cell.Biol.111:1593-1604 (1990)] of the α of integrin family subunit.The design of primers and the dog α that manages to increase _TM1Sequence

According to the N-terminal sequence information, three oligonucleotide probes that are used to hybridize have been designed: a) " Tommer ", the oligonucleotide of a complete degeneracy; B) " Patmer ", the oligonucleotide of a part degeneracy; C) " Guesser " nondegenerate oligonucleotide that uses based on the mammal codon.These probes are listed in following SEQ ID NO:6,7 and 8 respectively.Here and the nucleic acid symbol of other nucleotide sequences of this paper meet 37 C.F.R § 1.882.

5′-TTYAAYYTGGAYGTNGARGARCCNATGGTNTTYCA-3′ (SEQ?ID?NO：6)

5′-TTCAACCTGGACGTGGAGGAGCCCATGGTGTTCCAA-3′ (SEQ?ID?NO：7)

5′-TTCAACCTGGACGTNGAASANCCCATGGTCTTCCAA-3′ (SEQ?ID?NO：8)

Based on sequence data, (dog spleen CA)/peripheral blood macrophage cDNA library is hybridized and is not detected relevant clone for Stratagene, La Jolla to use these oligonucleotide to be cloned into λ ZAP_ to one under several low stringency conditions.

The phage library DNA that has designed purification in the dull and stereotyped lysate in the Stratagene library that four other oligonucleotide primers manage to introduce from above according to the N-terminal sequence of deriving subsequently is with pcr amplification dog α _TM1Sequence, these four primer called after 5 ' Deg, 5 ' Spec, 3 ' Deg and 3 ' Spec (list in SEQ ID NO:9,10,11 and 12 respectively, wherein Deg represents degeneracy, and Spec represents nondegenerate).

5′-TTYAAYYTNGAYGTNGARGARCC-3′ (SEQ?ID?NO：9)

5′-TTYAAYYTGGACGTNGAAGA-3′ (SEQ?ID?NO：10)

5′-TGRAANACCATNGGYTC-3′ (SEQ?ID?NO：11)

5′-TTGGAAGACCATNGGYTC-3′ (SEQ?ID?NO：12)

α _TM1Oligonucleotide primers and T3 or T7 carrier primer coupling, T3 and T7 primer are listed in SEQ ID NO:13 and SEQ ID NO:14 respectively, they can with the flanking sequence hybridization in polylinker zone in the Bluescript_ phasmid found among the λ ZAP_.

5′-ATTAACCCTCACTAAAG-3′ (SEQ?ID?NO：13)

5′-AATACGACTCACTATAG-3′ (SEQ?ID?NO：14)

Pcr amplification is containing magnesium and is containing Taq buffer (the Boehringer Mannheim of the Taq polymerase (Boehringer Mannheim) of 150ng library DNA, every kind of primer of 1 μ g, 200uMdNTPs and 2.5 units, indianapolis, IN) carry out in, product separates with 1% agarose gel electrophoresis in Tris-acetate-EDTA (TAE) buffer that contains 0.25 μ g/ml bromination second pyridine.DNA transfers to Hybond_ film (Amersham by absorbing to spend the night in 10 * SSPE, Arlington Heights, IL) on, after the transfer, the immobilized DNA 0.5M NaOH degeneration that contains 0.6M NaCl, with the Tris-HCl that contains 1.5M NaCl, pH8.0 neutralization, wash with 2 * SSPE before UV-crosslinked carrying out with Stratalinker (Stratagene) crosslinking apparatus.With film in prehybridization buffer (5 * SSPE, 4 * Denhardts, 0.8%SDS, 30% formaldehyde) 50 ℃ stirred incubation 2 hours.

Oligonucleotide probe 5 ' Deg, 5 ' Spec, 3 ' Deg and 3 ' Spec (being respectively SEQ ID NO:9,10,11 and 12) are with containing 100-300uCi γ P ³²In the Boehringer Mannheim kinase buffer liquid of the polynucleotide kinase of-dATP and 1-3 unit in 37 ℃ of labelling 1-3 hours.(NJ) chromatograph does not use 10mM Tris-HCl, pH8.0,1mM EDTA (TE) buffer to remove to the labelling of integrating for Pharmacia, Piscataway, and effluent directly adds prehybridization solution with Sephadex_G-25 fine.Film was handled 16 hours and was stirred and cyclic washing at 42 ℃ with probe, used 1 * SSPE/0.1%SDS to wash 15 minutes under 50 ℃ stringent condition at last.Trace subsequently on Kodak X-Omat AR film-80 ℃ the exposure 1-4 hour.

Oligonucleotide 5 ' Deg, 5 ' Spec, 3 ' Deg and 3 ' Spec only with use by oneself their as the hybridization of the PCR product of the reaction of primer, and can not as was expected with without they PCR product hybridization as the reaction of primer.Therefore, conclusion is that not having the PCR product is α _TM1Special because do not have product can with all suitable primer hybridizations.

Embodiment 3

Extensive affinity purification dog α _TM1Be used for inner order-checking

For being provided for other aminoacid sequence of design of primers, purification dog α _TM1Be used for inner order-checking.Prepare the albumen of inner order-checking with three parts of refrigerated spleens (every part about 50 gram) with from the frozen cell of two part spleens of adult dogs.50 gram spleens are used the homogenate of Waring agitator in the 200-300ml borate buffer.The homogenate material contains the buffer dilution of 4%NP-40 with 1 times of volume, then with mixture gentle agitation at least 1 hour.At a large amount of residues that 2000g removed in the lysate that is produced in centrifugal 20 minutes, (lysate further filters with Corning 0.4 microfiltration membrane system and makes it clarification for Corning, NY) prefilter or Corning 0.8 microstrainer filtration to use Corning then.

The Affigel_10 resin of the antibody coupling that spleen lysate and embodiment 2 introduce is combined into branch things such as 100ml by 150: 1 volume ratio, is incubated overnight 4 ℃ of rotations.Removed lysate in centrifugal 5 minutes at 1000g, be incubated overnight with the Affigel_10 mixed with resin of more antibody coupling and by above-mentioned.Then the branch things such as resin of absorption are merged and with the D-PBS/0.1%Tween_20 washing of 50 times of volumes, then resin is changed over to 50ml Biorad post.The albumen of absorption is with 0.1M glycine (pH2.5) eluting from resin of 3-5 times of volume; Collect the at different levels part of about 900 μ l, and neutralize, from each grade part, pipette 15 μ l, in isopyknic 2 * Laemmli sample buffer that contains 1/15 volume 1M dithiothreitol, DTT, boil with 100 μ l 1M Tris buffer, pH 8.0.With these samples at 8%Novex (San Diego, CA) electrophoresis on the polyacrylamide gel.(MA) program of recommending by manufacturer is dyed colour developing with coomassie dyestuff or silver for Enprotech, Natick to use the Daiichi test kit.To contain the proteic level of maximum part mixing and vacuum concentration.Rest solution is carried out 50% dilution with reproducibility Laemmli sample buffer, and goes up 1.5mm 7% polyacrylamide gel in Tris-glycine/SDS buffer.Use the Hoefer transfer device albumen to be transferred on the Immobilon film from gel by the program of introducing among the embodiment 2.

Downcut corresponding to dog α from 10 pvdf membranes _TM1Protein band, and obtained the total protein of 47 μ g, with band decolouring 5 minutes in the methanol of 4ml 50%, air drying, and be cut into the small pieces of 1 * 2mm.The film small pieces are immersed 2ml 95% acetone, 4 ℃, 30 minutes, and concussion, air drying then frequently.

Before membrane-bound albumen was carried out protein cleavage, (Pierce, Rockford IL) were dissolved in the formic acid of 1.25ml 70% with the Bromine cyanide. (CNBr) of 3mg.Then this solution is added and contain in the test tube of pvdf membrane small pieces, in the dark the room temperature incubation is 24 hours.Then supernatant (S1) is moved into another test tube, with the formic acid washing film small pieces of 0.25ml 70%.(S2) shifts out with this supernatant, adds in the supernatant (S1) of front.In blended supernatant (S1 and S2), add 2 milliliters of Milli Q water, then with the solution lyophilizing.The pvdf membrane small pieces are dry in nitrogen, with the acetonitrile of 1.25ml 60%, 0.1% tetrafluoro acetic acid (TFA) 42 ℃ of extractings 17 hours again.(S3) shifts out with supernatant, the film small pieces with the acetonitrile of 1.0ml 80%, 0.08% TFA 42 ℃ of extractings 1 hour again.This supernatant is mixed vacuum drying with the supernatant (S1, S2 and S3) of front.

Then exsiccant CNBr fragment is dissolved in 63 μ l 8M carbamide, 0.4M NH ₄HCO ₃Fragment is reduced in 5 μ l 45mM dithiothreitol, DTTs (DTT), and then 50 ℃ of incubations 15 minutes.Then solution is cooled to room temperature, and (Sigma, St Louis is MO) with the fragment alkaline lysis to add 5 μ l 100mM iodoacetamides.After 15 minutes, it is 2.0M that sample is diluted to the carbamide final concentration with 187 μ l Milli Q water at the room temperature incubation.Then in enzyme and albumen 1: 25 (w: ratio w) adds trypsin Worthington, Freehold, NJ), albumen was 37 ℃ of digestion 24 hours.Add 30 μ l TFA and stop digestion.

Then at the Waters 625 (Millipore of LC system, Milford MA) upward separates protein fragments with high performance liquid chromatography (HPLC), uses at 0.05%TFA and the equilibrated 2.1 * 250mm 5micron of HPLC water (buffer A) Vydac C-18 post (Vydac, Hesperia, MA).Peptide carries out eluting with ever-increasing 80% acetonitrile of concentration among the 0.04%TFA (buffer B), the buffer B eluting of 38-75% gradient 65-95 minute, the buffer B eluting of 75-98% 95-105 minute.Peptide is with 0.2ml/ minute flow velocity classification, and detects at 210nm.

After the classification, the aminoacid sequence of peptide is analyzed with the automatic edman degradation method.Sequence analysis uses the standard cycle of manufacturer to carry out on Applied Biosystems Model 437A protein sequencing instrument, and uses Model 610A Data Analysis software program version 1.2.1.All sequencing reagents are provided by Applied Biosystems.Below 7 aminoacid sequence was listed in 8 interior segments, wherein " X " expression aminoacid was uncertain.

VFQEXGAGFGQ (SEQ?ID?NO：15)

LYDXVAATGLXQPI (SEQ?ID?NO：16)

PLEYXDVIPQAE (SEQ?ID?NO：17)

FQEGFSXVLX (SEQ?ID?NO：18)

TSPTFIXMSQENVD (SEQ?ID?NO：19)

LVVGAPLEVVAVXQTGR (SEQ?ID?NO：20)

LDXKPXDTA (SEQ ID NO:21) design of primers

The internal amino acid sequence (listing in SEQ ID NO:22) of an acquisition is used to design the oligonucleotide primers of a complete degeneracy subsequently,, lists in SEQ IDNO:23 its called after p4 (R).

FGEQFSE (SEQ?ID?NO：22)

5′-RAANCCYTCYTGRAAACTYTC-3′ (SEQ?ID?NO：23)

Embodiment 4

A dog α _TM1Segmental PCR clone

Dog α _TM15 of gene ' part increases from the dog spleen cDNA of two strands with PCR.The dog spleen cDNA that preparation is double-stranded

1 gram is immersed in the liquid nitrogen on the dry ice from the frozen matter of pup spleen, 20mlRNA-Stat 60 buffer (Tel-Test B Inc, Friendswood, TX) in homogenate.Add the 4ml chloroform, solution is 12, and 000g carried out extracting in centrifugal 15 minutes.RNA precipitates from aqueous phase with 10ml ethanol.(Dynal, Oslo Norway) go up selection Poly A at Dynal Oligo dT Dynabeads_ then ⁺RNA.Dilute with total RNA merging such as 100 μ g of 5 things such as branch such as grade and with isopyknic 2X binding buffer liquid (20mM Tris-HCl, pH7.5,1.0M LiCl, 1mMEDTA, 0.1%SDS).Then with RNA with Oligo dT Dynabeads_ (all samples with 1.0ml or 5mg pearl) incubation 5 minutes.According to the program that manufacturer is recommended, using 2mM EDTA, pH7.5 eluting Poly A ⁺Before the mRNA, with the buffer washing pearl that contains 10mM Tris-HCl, pH7.5,0.15 M LiCl, 1mM EDTA and 0.1%SDS.Use the Poly A of eluting then ⁺MRNA and Boehringer Mannheim cDNA synthetic agent box are according to the program that manufacturer is recommended, the cDNA that preparation is double-stranded.

Part dog α _TM1The separation of cDNA

In the PCR of standard reaction, use oligonucleotide primers 5 ' Deg (SEQ ID NO:9) and p4 (R) (SEQ ID NO:23), use the Taq polymerase (BoehringerMannheim) of every kind of primer of double-stranded cDNA, the 500ng of 150ng, 200uM dNTPs and 1.5 units, in magniferous Taq buffer (Boehringer Mannheim), carry out.Product (1 μ l of the first set reaction) adding that obtains is taken turns in the reaction to increase product quantity with second of same primer.With this band eluting from 1% agarose gel, in the buffer of 10mMTris-HCl, pH8.0,1mM EDTA, 1.5mM NaCl, go to Schleicher﹠amp at 65 ℃; (Keene is NH) on the NA45 paper for Schuell.And the program of using TA clone's test kit (Invitrogen) to recommend according to manufacturer, be connected to pCR ^TmThe II carrier (Invirtogen, San Diego, CA) in.Connecting the mixture electricity is converted in the XL-1 Blue antibacterial (Stratagene).A clone 2.7 is determined and contains corresponding to α _TM1The sequence of peptide sequence, this sequence are not used in design of primers.

Order-checking Applied Biosystems 373A dna sequencing instrument (Foster City, CA), using two deoxidations to stop cycle sequencing test kit (ABI) carries out, in this test kit, fluorescently-labeled dNTPs mixes (McCabe an asymmetric PCR reaction as follows, " prepare single stranded DNA " with asymmetric PCR, " PCR method: methods and applications guide ", Innis etc. (editor) 76-83 page or leaf, Academic Press:New York (1990)).Sample was placed 4 minutes at 96 ℃, carried out 25 circulations of the following step then: 96 ℃, and 15 seconds, 50 ℃, 1 second, 60 ℃, 4 minutes.The sequencing data that comprises figure and text downloads in the sample file on the computer automatically.Whole sequence of inserting son of the clone 2.7 is listed in SEQ ID NO:24.

Manage (to introduce) the dog α of separation total length as embodiment 2 from the Stratagene library _TM1CDNA is success not.To clone 2.7 as probe, to use the hybridization of the low stringency condition of 30% formaldehyde, to about 1 * 10 ⁶Plaque screens, and does not obtain positive colony.Use from clone's specific oligonucleotide of 2.7 or with degenerate oligonucleotide (Tamura etc. based on conservative α subunit primitive GFFKR, above) the degenerate primer based on the aminoacid sequence of other fragments of peptides of coupling, manage to increase and clone the relevant downstream sequence of the sequence that shows in 2.7, not successful yet.

Embodiment 5

The dog α that infers _TM1The clone of people's congener

For managing to separate and dog α _TM1Homologous human sequence uses the about 1kb dog α from clone 2.7 _TM1Fragment is as probe.Probe uses NT2 (listing in SEQID NO:25) and p4 (R) (SEQ ID NO:23) primer under the condition that embodiment 2 introduces, be prepared with PCR.

5′-GTNTTYCARGARGAYGG-3′ (SEQ?ID?NO：25)

(Chatsworth, GA) Quick Spin test kit carries out purification by the program of manufacturer's recommendation to the PCR product with Qiagen.The program that the DNA of purification (200ng) recommends by manufacturer with Boehinger Mannheim random primer labelling test kit is used 200uCi α ³²P dCTP carries out labelling.Uncorporated isotope is removed with Sephadex_G25 (fine) gravity chromatograph.Probe is used 0.2N NaOH degeneration before use, and neutralizes with 0.4M Tris-HCl, pH8.0.

Preparation pCDNA/Amp (Invitrogen San Diego, CA) go up bacterium colony and offer thing by the Hybond_ filter paper (Amersham) in the people's spleen cDNA library in.Filter paper press earlier method degeneration and the neutralization that embodiment 2 introduces, follows in the prehybridization solution that contains 30% formaldehyde (8ml/ filter paper) 50 ℃ of incubations 2 hours, and gentle agitation.The label probe of as above introducing is added in this solution, and with filter paper 42 ℃ of incubations 14 hours.Filter paper in 2 * SSC/0.1%SDS 37 ℃ of washed twice, in 2 * SSC/0.1%SDS 50 ℃ of washed twice.Last strictness washing is a washed twice (1 * SSC is 150mM NaCl, 15mM sodium citrate, pH7.0) in 65 ℃, 1 * SSC/0.1%SDS, and filter paper exposed 6 hours on Kodak X-Omat AR film with intensifying screen.To offer to have on the thing bacterium colony of signal on magniferous LB culture medium (LBM)/Carbenicillin flat board, to rule duplicating, and be incubated overnight at 37 ℃.The line bacterium colony that is obtained is offered with Hybond_ filter paper, and these filter paper are handled as stated above.Filter paper under the stricter condition with labelling as stated above, from clone 2.7 the Methanamide hybridization solution of 1kb probe 50% in 50 ℃ of hybridization 3 hours.The filter paper that probe is handled washs with 0.1 * SSC/0.1%SDS, 65 ℃ final stringent condition, and exposes 2.5 hours on Kodak X-Omat AR film at-80 ℃ with intensifying screen.Identify positive colony, and in LBM/ Carbenicillin culture medium overnight incubation.The program of using Promega Wizard_miniprep test kit to recommend according to manufacturer prepares DNA by culture, and the DNA that is obtained is checked order.

Preliminary screening has obtained 18 clones, and under stricter hybridization conditions second take turns the screening produced a positive colony, with its called after 19A2.People α _dDNA sequence and the deduced amino acid of clone 19A2 are listed in SEQ ID 0:1 and 2 respectively.

People α _dThe feature of the polypeptide of cDNA and prediction

Clone 19A2 comprises whole coding regions of maturation protein, adds 5 ' stream signal sequence of 48 bases (16 amino acid residues) and 3 ' non-translated sequence of 241 bases, and this sequence does not stop with the sequence of polyadenylation.The core element amount of maturation protein is expected to be about 125kD.Its ectodomain estimates approximately to comprise 17 to 1108 amino acid residues of SEQ ID NO:2.This ectodomain and then one section stride homologous about 20 amino acid regions of diaphragm area (the residue 1109-1128 of SEQ ID NO:2) with people CD11c.Born of the same parents' intracellular domain contains 30 aminoacid of having an appointment (the approximately residue 1129 to 1161 of SEQ ID NO:2).This albumen also contain one with CD11a, CD11b and CD11c (Larson and Springer, above), α _E[Shaw, et al., J.Biol.Chem.269:6016-6025 (1994)] and VLA-1 and VLA-2 (Tamura etc., above) in common I (insert son) homologous about 202 the amino acid whose zones of domain (about residue 150 to 352).I domain in other integrins has proved and has participated in ICAM in conjunction with [Landis, et al., J.Cell.Biol.120:1519-1527 (1993); Diamond, et al., J.Cell.Biol.120:1031-1043 (1993)], this illustrates α _dAlso can combine with the member of the ICAM family of surface molecular.This zone does not also prove and is present in other integrin subunits.

α _dThe deduced amino acid demonstration and the sequence of CD11a, CD11b and CD11c 36%, 60% and 66% homology is arranged respectively approximately.Fig. 1 has shown CD11b (SEQ ID NO:3), CD11c (SEQ ID NO:4) and α _dThe aminoacid sequence comparison of (SEQ ID NO:2).

β ₂The cell intracellular domain of beta 2 integrin alpha subunit also has nothing in common with each other in same species usually, but indivedual α subunit also shows the homology of height between species.Consistent with these discoveries, α _dThe intracellular region territory different with CD11a, CD11b and CD11c significantly, except the verified affine GFFKR aminoacid sequence of film conservative in all alpha-6 integrins [Rojiani, et al., Biochemistry 30:98599866 (1991)].Because the cell intracellular domain of integrin relate to " inside and outside " signal conduction and affinity adjusting (Lands etc., above), so α _dMay with CD11a, CD11b and the interactional different cytoplasm interaction of molecules of CD11c, thereby participate in and other β ₂The signal pathway that integrin is different.

α _dExtracellular domain contain conservative and conservative DGSGS aminoacid sequence I domain adjacency, the DGSGS sequence is the necessary melts combine of ligand interaction zone [Michishita, et al.Cell 72:857-867 (1993)] in CD11b.Three extra supposition cation binding sites of CD11b and CD11c are at α _dGuarding in the sequence, is aminoacid 465-474,518-527 and 592-600 in clone 19A2 (SEQ ID NO:1).Its α _dThe homology of the respective regions of I domain and CD11a, CD11b and CD11c is respectively 36%, 62% and 57%.The low sequence homology in this zone shows α _dMay with one the cover with other known β ₂The different extracellular protein of the interactional albumen of integrin interacts.In addition, α _dTo other known β ₂The affinity of integrin ligands such as ICAM-1, ICAM-2 and/or ICAM-R, may with other β ₂The affinity difference (seeing embodiment 12) of proof during integrin/ICAM interacts.

Separate other people α _dThe cDNA clone is used for sequence to be proved

Be proof coding people α _dDNA sequence, separate other people cDNA in people's spleen Oligo dt-primed cDNA library (Invitrogen) of method from a pcDNA/Amp (introducing among the embodiment 5) with hybridization.This library is to make up by the cDNA of length on the selection agarose gel electrophoresis greater than 3kb.Hybridize used probe from the α that introduces below _d5 ' zone.Hybridization conditions and above-mentioned initial gross separation people α _dClone's condition is identical.Have only following hybridization post processing different, filter paper in 2 * SSC/0.1%SDS in room temperature washing twice, 42 ℃ of washings once at 2 * SSC/0.1%SDS.Filter paper spends the night with Kodak X-Omat AR exposure.

5 ' α _dHybridization probe carries out under PCR use primer CD11c 5 ' For (SEQ ID NO:94) and CD11c 5 ' Rev (the SEQ ID NO:95) condition below with PCR preparation from the 19A2 clone.Sample was placed 4 minutes at 94 ℃, carried out 30 circulations of the following step then: i) 94 ℃, and 15 seconds; Ii) 50 ℃, 30 seconds, iii) 72 ℃, 1 minute.Use the Perkin-Elmer9600 thermal cycler.

CD11c?5′For：(5′)CTGGTCTGGAGGTGCCTTCCTG(3′)?(SEQ?ID?NO：94)

CD11c?5′Rev：(5′)CCTGAGCAGGAGCACCTGGCC(3′)?(SEQ?ID?NO：95)

Amplified production uses BioRad, and (Hercules, CA) the Prep-A-Gene test kit carries out purification according to the program of manufacturer's recommendation.5 ' the α that is obtained _dProbe is that about 720 bases are long, corresponding to the 1121-1839 nucleotide zone of SEQ ID NO:1.(Indianapolis, Indiana) use by the program of manufacturer's recommendation by the random primer labelling test kit with Boehinger Mannheim for the DNA of purification (about 50ng) ³²PdCTP carries out labelling.Uncorporated isotope spins with Centrisep_, and (Princeton Separations, Adelphia NJ) remove according to the program that manufacturer is recommended post.The probe of labelling is added in the filter paper in the prehybridization solution that contains 45% Methanamide, spend the night 50 ℃ of incubation reaction.Behind the incubation, filter paper washs by top introduction.

13 are cloned in to duplicate and offer that signal is arranged on the thing.Positive colony is chosen from motherboard,, be laid on Hybond_ (Amersham) filter paper by different dilution factors in LBM and Carbenicillin (100 μ g/ml) dilution.The double film uses the solution identical with original cross to hybridize, and after hybridization, filter paper washs with 2 * SSC/0.1%SDS, 42 ℃ final stringent condition, and exposes on film.

First 13 positive colonies identifying have 10 to take turns in the screening second and to be proved.In these 10 clones, 2 clones (called after A7.Q and A8.Q) are checked order, and determine its coding people α _dClone A7.Q is found and is about 2.5kb, comprises 5 ' untranslated region of 5 ' targeting sequencing, part coding region and extra 60 bases.It is the result of intron zone errors montage who is equivalent to 2152 nucleotide of SEQ ID NO:1 that this incomplete coding region is determined.A8.Q is determined and is about 4kb, crosses over whole α d coding region, also contains the intron zone of 305 bases that are equivalent to SEQ ID NO:1.With isolating α first _dClone (SEQ ID NO:1) relatively has a difference to be found, and promptly A7.Q and A8.Q all are defined in base 1495 places has one three base CAG codon to insert.The sequence of clone A7.Q and A8.Q is listed in SEQID NO:96 and 97 respectively, lists in SEQ ID NO:98 and 99 respectively by the human sequence and the aminoacid sequence of deriving accordingly that clone A7.Q and A8.Q edit.

Embodiment 6

People α in the tissue _dThe Northern that expresses analyzes

For measuring α _dRelative expression's level and tissue specificity use from the fragment of clone 19A2 and carry out the Northern analysis as probe.Will be from the total RNA of about 10 μ g of each one tissue or cultured cell system formaldehyde agarose gel under the situation that has 1 μ g bromination second pyridine.After 4 hours, RNA is transferred to nitrocellulose membrane (the Schleicher ﹠amp of soaked overnight in 10 * SSC at the 100V electrophoresis; Schuell) on.With film in a vacuum 80 ℃ the baking 1.5 hours.With the prehybridization solution of the 3-that contains 50% Methanamide (N-morpholino) propane sulfonic acid (MOPS) 42 ℃ of closing membranes 3 hours.The fragment of clone 19A2 is used the indication of Boehringer Mannheim random primer test kit according to manufacturer, uses α P ³²DCTP and α P ³²Both carry out labelling dTTP.Remove with the Sephadex_G25 post in the uncorporated TE of the being marked at buffer.Film is by 1.5 * 10 ⁶The amount of/ml prehybridization buffer is handled with probe.Then with trace room temperature with 2 * SSC/0.1%SDS, 42 ℃ with 2 * SSC/0.1%SDS, 50 ℃ with 2 * SSC/0.1%SDS, 50 ℃ with 1 * SSC/0.1%SDS, 50 ℃ with 0.5 * SSC/0.1%SDS, at 50 ℃ with 0.1 * SSC/0.1%SDS continuous washing.Then trace is used exposure 19 hours.

Use discloses from the hybridization of the BstXI fragment (being equivalent to 2011 to 3388 nucleotide among the SEQ ID NO:1) of clone 19A2: in about 5kb scope weak signal is arranged in the total RNA of liver, Placenta Hominis, thymus and tonsil; In kidney, brain or heart sample, do not find signal.As measuring with bromination second pyridine dyeing, the RNA amount that exists in the kidney sample band is minimum.

When using second fragment (base 500 to 2100 zones that comprise SEQ ID NO:1) of clone 19A2, using polyA ⁺The people of RNA (Clontech) organizes the rna transcription thing that detects two kinds of different sizes in Northern (MTN) trace more.In spleen and skeletal muscle, observe the band of about 6.5kb, in lung and peripheral blood leucocyte, observe the band of 4.5kb.Variation on the observed size may be that underlying cause causes: tissue-specific adenosine acidify, probe and other integrins family member's cross reaction or hybridize with alternatively spliced mRNA.

The Northern that use is carried out from the 3rd fragment (crossing over 2000 to 3100 nucleotide among the SEQ ID NO:1) of clone 19A2 analyzes with consistent with the segmental result of other clone 19A2.

From the RNA of three kinds of bone marrow pedigree cell lines also with being equivalent to the nucleotide 500 to 2100 of SEQ ID NO:1 and 2000 to 3100 fragment is analyzed as probe.The THP-1 cell that stimulates with PMA has provided the signal (about 5.0kb) that spreads in same magnitude range in advance, and intensity is slightly larger than tissue signal.From not stimulating and the RNA and the α of the HL-60 cell of DMSO stimulation _dProbe hybridization provides the signal with the tissue sample same intensity.As if but PMA has improved signal intensity.Because PMA and DMSO order about the HL-60 cell respectively to Monocytes and the differentiation of granulocyte approach.This results suggest, α in the Monocytes type _dExpression strengthen.U937 cellular expression α _dInformation, but this signal does not increase because PMA stimulates.In Molt, Daudi, H9, JY or Jurkat cell, do not detect band.

Embodiment 7

People α _dThe transient expression of construct

The people clones 19A2 and lacks an initial methionine codon and some possible 5 ' signal sequence.Therefore, for preparation contains people's expression plasmid of 19A2 sequence, we have adopted two kinds of strategies.First kind of strategy made up two kinds of plasmids, in two kinds of plasmids, come own coding CD11b or CD11c gene the signal peptide sequence montage to clone 19A2 in, prepared a kind of chimeric α _dSequence.Second method has made up the third plasmid, has wherein added the adenylic acid base initial methionine of encoding in 0 position of clone 19A2.

These three plasmids contain coding for alpha _dSequence or chimeric α _dThe zones of different of 5 of sequence ' part.α _dThe zone is with species specific 3 ' primer BamRev (listing in following SEQ ID NO:26) and three kinds of 5 ' primers is a kind of as primer, increases with PCR (seeing the condition of embodiment 2).Three kinds of 5 ' primers contain in sequence: (1) is in the identical non-specific base, the EcoRI site of position 7-12, the total Kozak sequence of position 13-18 of position 1-6 for ease of digestion; (2) the part signal sequence of CD11b (primer ER1B) or CD11c (primer ER1C), an or adenylic acid (primer ER1D); (3) other 15-17 base of 5 ' sequence of 19A2 cloned in covering specifically, is used to make primer annealing.Primer ER1B, ER1C and ER1D list in SEQ ID NO:27,28 or 29 respectively.Initial methionine codon underscore wherein, the following stroke two-wire in EcoRI site.

5′-CCACTGTCAGGATGCCCGTG-3′ (SEQ?ID?NO：26)

(SEQ?ID?NO：27)

(SEQ?ID?NO：28)

(SEQ?ID?NO：29)

The PCR product that is obtained digests with EcoRI and BamHI.

All three kinds of plasmids all contain a common α _dRegional (and then inserting the 5 ' regional downstream that a last joint is introduced) comprises 3 ' end that α clones.Extend to the 2nd α in the XbaI site in carrier 3 ' polylinker zone of cloning 19A2 from nucleotide 625 _dSeparate with XbaI digestion clone 19A2 with BamHI in the zone.

Prepare coupled reaction, wherein 3 ' α _dBamHI/XbaI fragment and three kinds of 5 ' α _dOne of EcoRI/BamHI fragment connects, and uses Boehringer Mannheim to connect buffer and T4 ligase (every reaction 1 unit).After 4 hours, in each reaction, add usefulness EcoRI and the carrier pcDNA.3 (Invitrogen) of XbaI digestion and the ligase of other 1 unit of right quantity at 14 ℃ of incubations.Continue reaction 14 hours.Use 1/10th reactant mixture conversion XL-1 Blue competent cell then.Cultivate the clone who is obtained, and press embodiment 5 DNA isolation.Digest with EcoRI and to identify that three these restriction enzyme site positives are the male clone of processing signal sequence.With its called after pATM.B1 (CD11b/ α _d, from primer ER1B), pATM.C10 (CD11c/ α _d, from primer ER1C) and pATM.D2 (adenylic acid/α _d, from primer ER1d).The existence of proper signal sequence confirms with nucleic acid sequencing among each clone.

α discussed above _dThe expression of plasmid is with every kind of plasmid and a CD18 expression plasmid--pRC.CD18 cotransfection COS cell carries out.As positive control, the COS cell is also used plasmid pRC.CD18 and a CD11a expression plasmid--pDC.CD11A cotransfection.

Before 16 hours, cell is added culture medium (DMEM/10%FBS/penstrep) in transfection, in the culture dish of handling by the 50% Corning tissue culture that converges the 10cm that packs into.To following all methods, from ware, do not take out cell with not containing tryptic Versene buffer (PBS that contains 0.5mM NaEDTA).Before transfection, plate washs once with the DMEM of serum-free.Every kind of plasmid with 15 micrograms in each plate adds in the 5ml transfection buffer (DMEM that contains 20 μ g/ml DEAE-Dextran and 0.5mM chloroquine), and after 1.5 hours, cell was suffered a shock 1 minute with 5ml DMEM/10%DMSO at 37 ℃ of incubations.Replace this DMSO solution with culture medium by the every plate of 10ml/ then.

The transfectant that is obtained is analyzed with ELISA, FACS and immunoprecipitation by embodiment 8,9 and 10 methods of introducing.

Embodiment 8

The elisa assay of COS transfectant

For determining CD18 expression plasmid pRC.CD18 and α _dWhether the COS cell of expression plasmid cotransfection is at its cell surface expression and the bonded α of CD18 _d, use the first antibody that produces at the CD18 TS1/18 of ATCC HB203 purification (for example by) to carry out PCR.As positive control, also to cotransfection CD18 expression plasmid and a kind of CD11a expression plasmid--the cell of pDC.CD11A carries out ELISA.In this contrast, first antibody comprises CD18 antibody and anti-CD11a the antibody TS1/18 of ATCC HB202 purification (for example, by).

For carrying out ELISA, shift out with the Versene buffer from the cell of each plate, and change a Corning tissue culturing plate that 96 independent holes are flat over to.Before test with cell incubation 2 days in culture medium.Then plate is pressed twice of D-PBS/0.5% bony fish skin gelatin (Sigma) solution washing in 150 μ l/ holes.Except that process color, this buffer is used for all steps.All washings and incubation all carry out in room temperature.The hole was sealed 1 hour with gelatin solution, and first antibody is diluted to 10 μ g/ml with gelatin solution, added 50 μ l then in every hole.Each first antibody is done three multiple holes.Behind the incubation 1 hour, plate washs 3 times with gelatin solution by 150 μ l/ holes.(sheep anti mouse Ig/HRP-Fc specificity (Jackson, West Grove, PA)) adds in the entering plate by 50 μ l/ holes, and incubation 1 hour for dilution in 1: 3500 two anti-.After washing three times, plate adds 15% sulphuric acid in 50 μ l/ holes then with (Sigma) solution (the 1mg/ml OPD in the citrate buffer solution) colour developing 20 minutes of the o-phenylenediamine (OPD) in 100 μ l/ holes.

The result who with anti-CD18 specific antibody transfectant is analyzed in the ELISA program shows, only do not having obvious expression with respect to background in the cell with the plasmid transfection of the CD18 that encodes.With the special antibody of CD18 or with the special reagent analysis of CD11a the time, show the expression higher with the cell of the plasmid co-transfection that contains CD11a and CD18 than background.Further to coding CD18 and a kind of α _dThe cell of expression construct (pATM.C10 or pATM.D12) cotransfection the analysis showed that the expression of CD18 is by α _dExpression simultaneously recovers.In transfection in the COS cell of pATM.C10 or pATM.D12, the raising that detected CD18 expresses can be compared with observed raising in cotransfection CD11a/CD18 positive control cell.

Embodiment 9

The facs analysis of COS transfectant

For carrying out facs analysis, the cell in the culture dish is raised with fresh culture medium second day of transfection, and before testing incubation 2 days.Transfectional cell washs once with 5ml FACS buffer (DMEM/2%FBS/0.2% Hydrazoic acid,sodium salt) with pipetting in the 3ml Versene slave plate, and with being diluted to 500,000 cells/0.1mlFACS buffer sample.The link coupled Mus 23F2G of CFSE (anti--CD18) (the ATCC HB11081) that in each sample, adds special antibody (Becton Dickinson) of the link coupled CD18 of 10 microlitre 1mg/ml FITC, CD11a or CD11b or 800 μ g/ml.Then with sample incubation on ice 45 minutes, with the FACS buffer by each 5ml washing 3 times.And be suspended in again in the 0.2ml FACS buffer.Then with Becton Dickinson FACscan on the sample, and with Lysys II software (BectonDickinson) analytical data.

The COS cell of CD18 sequence transfection is not only to CD18, CD11a or CD11b dyeing.During with the CD11a/CD18 cotransfection, about 15% cell is by the antibody staining at CD11a or CD18.All transfections CD18 and any α _dThe cell of construct can not produce CD11a and CD11b can detectedly be dyeed.PATM.B1, pATM.C10 and pATM.D12 group can dye 4%, 13% and 8% the positive to CD18 respectively.The fluorescence of the positive colony in the CD11a/CD18 group is higher four times than background.Compare with it, use α _dThe colony that construct and CD18 construct cotransfection produce shows than background increases 4-7 fluorescence intensity doubly.

Embodiment 10

The people α of the COS cell of cotransfection _dThe biotin labeled immunoprecipitation of/CD18 complex

Managed to cotransfection the α that introduces respectively among CD18 and the every kind of embodiment 7 _dThe cell of expression plasmid carries out immunoprecipitation, determines α _dWhether can separate as the part of the distinctive α β of integrin heterodimer complex.

Transfectional cell (1-3 * 10 ⁸Cell/group) pipette from culture dish with the Versene buffer, washing is 3 times in 50ml/ group D-PBS.(Pierce, Rockford is IL) room temperature labelling 15 minutes with the Sulpho-NHS biotin of 2mg for each sample.React to wash among the cold D-PBS that is used in the 50ml/ sample and carry out cancellation 3 times.The cell of washing is suspended in 1ml lysis buffer (1%NP40,50mM Tris-HCl, pH8.0,0.2M NaCl, 2mM Ca again ⁺⁺, 2mM Mg ⁺⁺And protease inhibitor) in, and incubation on ice 15 minutes.Made the insoluble substance precipitation in centrifugal 5 minutes 100,000, supernatant is moved into a new pipe.For the material of removal, carry out a pre cleaning step earlier with the rat immune globulin nonspecific reaction.With the rat immune globulin of 25 micrograms (Cappel, West Chester, PA) with supernatant at 4 ℃ of incubations.2.5 after hour, (by protein A Sepharose_4B and rabbit anti-mouse igg preparation, all from Zymed, San Francisco CA), continues to rotate incubations 16 hours at 4 ℃ the link coupled Sepharose_ of the anti-Mus Ig of rabbit of adding 100 μ l (25 μ g) in each sample.Centrifugally from supernatant, shift out the Sepharose pearl.Behind the pre cleaning, subsequently supernatant was handled 2 hours at 4 ℃ with the anti-CD18 antibody of 20 μ g (TS1.18).Come separation antibody/antigenic compound from supernatant by the anti-Mus/protein A of the rabbit of introducing above-Sepharose_ prepared product incubation with 100 μ l/ samples.Pearl is washed 4 times with 10mM HEPES, 0.2M NaCl and 1%Triton-X100_.With the pearl precipitation of washing, and in containing 2 * Laemmli sample buffer of 2% mercaptoethanol, boiled 10 minutes 20 μ l.Sample is centrifugal, and last 8% good in advance Novex polyacrylamide gel (Novex), 100V electrophoresis 30 minutes used.Albumen shifted 1 hour at 200mAmps in the TBS-T buffer, transferred to (Schleicher﹠amp on the nitrocellulose membrane; Schuell).Film was handled 1 hour with the streptavidin horseradish peroxidase (BOD) (Boehringer Mannheim) of dilution in 1: 6000, then washed 3 times in TBS-T.Use the indicated number trace of Amersham enhanced chemiluminescence test kit then according to manufacturer.Then film is gone up exposure 0.5 to 2 minute at Hyperfilm MP (Amersham).

The immunoprecipitation of the CD18 complex of pRC.CD18 and pATM.B1, pATM.C10 or the pATM.D12 cells transfected of using by oneself has shown a kind of surface expression of heterodimer, this heterodimer contain the 100kD that has an appointment, with the prediction CD18 β chain of the same size and corresponding to α _d, the α chain of about 150kD.

Embodiment 11

People α _dStable transfection in Chinese hamster ovary cell

For determining α _dWhether be combined into heterodimer with CD18 and be expressed in cell surface, all instantaneous and transfection stably of the cDNA of every the chain of encoding is to lacking α _dIn the cell line of CD18.

For carrying out these tests, with α _dCDNA increases with the extra targeting sequencing of introducing as embodiment 7 and a Kozak consensus sequence, and sub-clone is in expression vector pcDNA3.Last construct called after pATM.D12, with the coding people CD18 of itself and a modification commercial carrier--the pDC1.CD18 cotransfection is to dihydrofolate reductase (DHFR) ^-Chinese hamster ovary (CHO) cell in.The plasmid pDC1.CD18 DHFR that encodes ⁺Labelling, transfectant can be selected with the anacid culture medium of suitable nucleoside, and the modification that produces pDC1.CD18 is as follows:

Plasmid pRC/CMV (Invitrogen) is a mammalian expression vector that has cytomegalovirus promoter and amicillin resistance marker gene.To be inserted into 5 of pRC/CMV SV40 replication origin ' end from the DHFR gene of plasmid pSC1190-DHFR.In addition, will be connected to 3 ' end of DHFR gene in the pRC/CMV/DHFR construct from the polylinker in 5 of plasmid pHF2G-DHF ' zone.Then the CD18 coded sequence is cloned between the 5 ' flank polylinker zone and bovine growth hormone poly A coding region of plasmid of generation.

The surface expression of CD18 uses monoclonal antibody TS1/18 to analyze with flow cytometry.Detected α in this cell line _dAnd the immunoprecipitation of making of the transient expression in the COS cell among the formation of the heterodimer between the CD18 and the embodiment 10 is consistent.

Embodiment 12

People α _dThe mode that relies on CD18 combines with ICAM-R

Use for reference the leukocyte integrin of proof energy mediated cell cells contacting and the interactional report [Hynes, Cell 69:11-25 (1992)] of ICAIU (ICAMs), the α of expressing cho cell _dThe bonded ability of/CD18 and ICAM-1, ICAM-R or VCAM-1 is estimated with two kinds of methods.

In same test, ICAM-1, ICAM-R or the VCAM-1 IgG1 fusion rotein of solubility is immobilized on the plastics, and measures α _d/ CD18 CHO transfectional cell and the bonded ability of immobilized part.Transfectional cell calcein inner marker, with binding buffer liquid (RPMI that contains 1%BSA) washing, and only at buffer (containing or do not contain the PMA of 10ng/ml) or contain incubation in the buffer of anti-CD18 monoclonal antibody of 10 μ g/ml.Transfectional cell is added in the 96 hole Immulon_4 titer plate that ICAM-1/IgG1, ICAM-R/IgG1 or VCAM-1/IgG1 fusion rotein with solubility wrap quilt in advance, or be used as negative control with the bovine serum albumin bag.The series number that the design of the soluble form of these adhesion molecules has been submitted in 5 days Augusts in 1993 submitting to jointly and own together is No.08/102, introduces in 852 the U.S. Patent application with fully open.Before the cell that adds labelling, the hole is sealed with the PBS that contains 1%BSA.Plate immersed among the PBS contain 0.1%BSA washed in 20 minutes after, (Millipore, Milford MA) measure remaining total fluorescence in every hole to use Cytofluor_2300.

In the test of using immobilized ICAMs, α _dThe cell of/CD18 cotransfection all show the hole than BSA bag quilt increase 3-5 doubly with the combining of ICAM-R/IgG1 hole.This bonded specificity and CD18 dependency prove with the inhibitory action of anti-CD18 antibody TS1/18.Combining of CD11a/CD18 cells transfected and ICAM-1/IgG1 hole, CD11a/CD18 transfectional cell with observed it is suitable with combining of the hole of BSA bag quilt only after anticipating, just show with PMA with the ICAM-1/IgG1 hole be combined with 2-3 raising doubly, α _d/ CD18 cells transfected is handled with PMA does not influence itself and the combining of ICAM-1/IgG1 hole.Do not detect α _d/ CD18 transfectional cell combines with the VCAM-1/IgG1 hole.

α _d/ CD18 transfectional cell is measured with ICAM-1/IgG1, ICAM-R/IgG1 or the combining with flow cytometry of VCAM-1/IgG1 fusion rotein of solubility.In each is measured, with about 100 ten thousand α _d/ CD18 transfection CHO cell (grow in to shake and carry out high expressed in the bottle) is suspended in the 100 μ l binding buffer liquid (RPMI and 1%BSA) that contain or do not contain the anti-CD18 antibody of 10 μ g/ml.After 20 minutes, cell washs in binding buffer liquid at the room temperature incubation, and adds ICAM-1/IgG1 or ICAM-R/IgG1 fusion rotein to final concentration 5 μ g/ml.Be combined in 37 ℃ and carried out 30 minutes,, and be suspended in the binding buffer liquid that 100 μ l contain the link coupled goat anti-human igg 1 of FITC of dilution in 1: 100 again then with cell washing 3 times.Behind the incubation 30 minutes,, and be suspended in 200 μ l binding buffer liquid, analyze with BectonDickinson FACScan with sample washing 3 times.

The α of about 40-50% _d/ CD18 transfectional cell shows and to combine with ICAM-R/IgG1, but not with ICAM-R/IgG1 or VCAM-1/IgG1 protein binding.Anticipating transfectional cell with PMA does not influence itself and the combining of ICAM-1/IgG1, ICAM-1/IgG1 or VCAM-1/IgG1, and this is consistent with the immobilization adherence test.Handle α with being combined in of ICAM-R with anti-CD18 antibody TS1/18 _dBe reduced to background level behind the/CD18 transfectional cell.

Measure the data declaration of collecting, α by these double combination _d/ CD18 combines with ICAM-R, and compares more preferably with the combination of ICAM-1 and VCAM-1.α _d/ CD18 and the binding ratio of ICAM-R and combining more preferably of ICAM-1 are with opposite with the observed result of CD11b/CD18 with CD11a/CD18.Therefore, α _d/ CD18 is bonded to be modified with the immunologic function that hope optionally influences the normal and pathology that ICAM-R plays a major role.And, at the immunologic opsonin antibody inhibition ICAM-R and the α that detect at the different ectodomains of ICAM-R _dIn the similar test of the bonded ability of/CD18 transfectional cell, the result shows, α _dThe different structure territory of/CD18 and CD11a/CD18 and ICAM-R interacts.

CD11a/CD18 can not combine explanation with ICAM-1/IgG1 or the ICAM-R/IgG1 in the solution, and the binding affinity between CD11a/CD18 and ICAM1 or the ICAM-R is too low, can not combination in solution.And detect α _d/ CD18 then shows very high binding affinity with combining of ICAM-R/IgG1.

In the test of above-mentioned introduction, the VCAM-1/Ig fusion rotein contains 7 extracellular immunoglobulin like domain.Fusion rotein prepares in the Chinese hamster ovary celI of transfection, and protein yield is measured with ELISA.From the not purified direct use of 7 domain VCAM-1 fusion rotein of Chinese hamster ovary celI supernatant, it is extremely low that protein yield is found.Because the low protein yield in the VCAM-1 prepared product uses a kind of business-like VCAM-1 prepared product (R ﹠amp; Systems, Minneapolis MN) detects α again _d/ CD18 combines with VCAM-1's, to determine whether being that the VCAM-1 of low concentration has caused not detecting α _dCombination.

As before, the α of expressing cho cell _dBe used for adopting the adherence test of immobilized recombinant paramyxovirus attached molecule with CD18.Use flow cytometry, the result shows, α _dThe expressing cho cell α of transfection _dWith CD18, and do not express other β ₂Integrin.Find also that simultaneously the Chinese hamster ovary celI of transfection is not expressed two kinds of bonded ligandins of known VCAM-1--α ₄β ₁And α ₄β ₇Parent's Chinese hamster ovary celI system shows not express alpha ₄Or β ₂Integrin.Basically undertaken by top introduction in conjunction with measuring.

The result shows, α _dThe Chinese hamster ovary celI of transfection and immobilized VCAM-1 combine with about 14.2% ratio, compare with it, and it combines with 7.5% ratio with immobilized BSA, selects plain ratio with 2.8% to combine with immobilized E-.In addition, when the monoclonal antibody that exists at first domain of VCAM-1, be blocked (3.0% in conjunction with) with combining basically of immobilization VCAM-1.Parent's Chinese hamster ovary celI does not select element or BSA to combine (all hanging down and 2% in conjunction with ratio) with VCAM-1, E-.After going down to posterity, also can reducing of the Chinese hamster ovary celI of transfection through the series of cell, this with through observed α after the same time _dSurface expression reduces consistent.

For determining the nature express alpha _dThe cell of/CD18 whether with VCAM-1 as binding partners, separate the eosinophilic granulocyte of peripheral blood, under the condition that has 10ng/ml IL-5, cultivate 5-7 days to improve α _dExpression.Flow cytometry shows that IL-5 makes α _dExpression improve 2-4 doubly, but to α ₄Not effect of expression.

The result shows, the eosinophilic granulocyte of cultivation and immobilized VCAM-1 combine with 28.8% ratio, and in conjunction with by a kind of anti-CD18 monoclonal antibody (combination rate is 17.1%) and a kind of at α ₄Monoclonal antibody (combination rate is 18.1%) part suppress.Opposite with the PRELIMINARY RESULTS of low-level and/or impure VCAM-1 with the front, these data show α _dβ ₂It is a kind of part of VCAM-1.

The FACS adherence test of introducing above is used to detect a kind of ICAM-R mutant E37A/Ig and express alpha _dThe combination of the Chinese hamster ovary celI of/CD18.E37A/Ig shown got rid of with the LFA-1/Ig chimera in conjunction with (Sadhu etc. " cell adhesion with communicate by letter " 2 volumes, 429-440 page or leaf (1994)).Mutain from the Chinese hamster ovary celI of stable transfection system is expressed with soluble form, and presses Sadhu etc., and the Prosep_A column purification is used in introduction above.

In repeated in experiments, do not detect E37A/Ig and α _dThe combination of/CD18 transfectional cell.The chimeric average fluorescent strength of E37A/Ig (MFI) that arrives with the link coupled anti-people's antibody test of FITC is identical with the MFI of independent detection antibody, and this explanation uses in test E37A/Ig not detect the signal higher than background.Similarly, in the ELISA that is undertaken by the introduction of embodiment 14, the E37A/Ig mutant does not show and immobilized α _d/ CD18 coupling.α _dCombine with iC3b

Complement component C3 can be formed complex iC3b by the protease cutting, and iC3b starts the alternative route of complement activation, the cell-mediated destruction that finally causes target.CD11b and CD11c participate in the iC3b combination, and participate in the particulate macrophage phagocytic of iC3b bag quilt subsequently.A fragments of peptides in the CD11bI domain is verified recently to be the interactional site of iC3b [Ueda, et al., Proc.Natl.Acad.Sci. (USA) 91:10680-10684 (1994)].The calmodulin binding domain CaM of iC3b is at CD11b, CD11c and α _dHigh conservative, this has pointed out a kind of α _d/ iC3b binding interactions.

α _dWith combining of iC3b with transfectional cell or natural express alpha _dThe sheep red blood cell (SRBC) (sRBC) of cell line (for example, PMA stimulate HL60 cell) and iC3b bag quilt in a rosette, carry out.[Dana，et?al.，J.Clin.Invest.73：153-159(1984)]。α _dThe HL60 cell (positive control) that/CD18 CHO transfectional cell, VLA4-CHO transfectional cell (negative control) and PMA stimulate forms rosettes under the situation of existence and not anti-CD18 monoclonal antibody (for example TS1/18.1) ability compares.

Embodiment 13

Use flicker to get close to the mensuration screening and identify α _dBonded regulator

α of the present invention _dPart and its binding partners (α _dPart/anti-α _dPart to) between bonded specificity mortifier, can determine with several different methods, for example mainly be illustrated in U.S. Patent No. 4,271,139, Hart and Greenwald, Mol.Immunol.12:265267 (1979), and Hart and Greenwald, determination techniques is got close in the flicker of J.Nuc.Med.20:1062-1065 (1979).Above-mentioned document all draws at this and is reference.

In brief, with α _dPart/anti-α _dA right member of part directly or indirectly is combined on the solid support.Catch indirectly and relate to a kind of direct and bonded monoclonal antibody of holder, a specificity epitope of the C-terminal of the integrin β catenin of this antibody capable identification solubility.This epi-position can be hemagglutinin or mycobacteria III9 epi-position [Anderson, etal., J.Immunol.141:607-613 (1988)].A kind of fluorometric reagent also combines with holder.In addition, fluorometric reagent can be by U.S. Patent No. 4,568, and 649 introduction is incorporated in the holder, and this patent is drawn at this and is reference.α _dPart/anti-α _dThe non-holder binding members radioactive compound labelling that part is right, the ray of this radioactive compound emission energy fluorescence excitation reagent.When part combined with radiolabeled anti-part, the bonded fluorometric reagent of label and holder was enough approaching, fluorescence excitation reagent, and produce light emission.When not in conjunction with the time, label general with solid support too far and can not fluorescence excitation reagent, light emission is very weak.Measure the light of emission, this emission light is relevant with the combination of part and antibody ligand.In sample, add binding inhibitor and will reduce fluorescent emission, because mortifier stops radioactive label to be captured nearby by solid support.Therefore, binding inhibitor can be identified the influence of the fluorescent emission in the sample by them.Potential α _dAnti-part also can identify with similar methods.

Use soluble recombining α as follows _d/ CD18 leucine zipper structure (seeing embodiment 14) is got close to the bonded regulator of screening CAM in the mensuration in flicker.With anti-α subunit of non-barrier or the immobilization of anti-β subunit antibody that is coated in advance on the scintillator embedding plate of reorganization integrin.In plate, add a chemical library chemical compound and a species specific biotin labeled CAM/Ig chimera simultaneously.CAM/Ig is chimeric to be detected in conjunction with the streptavidin with labelling.In this test, (long-chain, Pierce) program of recommending according to manufacturer is carried out biotin labeling with NHS-Sulfo-biotin LC for ICAM-1/Ig and ICAM-R/Ig.Labelled protein in ELISA reaction, with streptavidin-HRP and when then detecting with the OPD colour developing, still can with the reaction of CAM specific antibody, and can show and immobilized LFA-1 reacts.

In addition, will recombinate leucine zipper protein purification or partial purification, direct coated is on the flicker embedding plate.Add unlabelled CAM/Ig chimera and chemical library chemical compound simultaneously.Bonded CAM/Ig uses ¹²⁵The anti-people Ig of I labelling detects.

The another kind of selection be, with the CAM/Ig proteopexyization of purification on the flicker plate.The concentrated supernatant that in plate, adds the cell of chemical library chemical compound and express recombinant leucine zipper integrin.The combination of reorganization integrin detects with anti-α subunit of barrier a kind of labelling, non-or anti-β subunit antibody.

Screen micromolecular regulator

Get close to a kind of of mensuration as flicker and substitute identical α _dBinding partners and inhibitor can be tested with the ELISA sample of introducing below and identify.

The α of solubility _d/ CD18 leucine zipper (LZ) structure (seeing embodiment 14) is used anti-α _dAntibody 212D (seeing embodiment 15) catches from tissue culture's supernatant.212D antibody is cushioned liquid (pH9.5) with the bicarbonate bag and spends the night at 4 ℃ and be fixed in 96 hole Immulon_IV plates (Costar).Same method is the anti-CD11a antibody of being used for fixing TS2/4.1 also, a kind of LFA-1 leucine zipper of being used for fixing (LFA-1LZ) fusion rotein, LFA-1 test VCAM-1 in conjunction with the time as negative control, test ICAM-1 in conjunction with the time as positive control.Plate sealed 1 hour with 3% bovine serum albumin in 300 μ l/ holes, and washed with D-PBS.Add express alpha by 100 μ l/ holes _dTissue culture's supernatant of the stable CHO transfectional cell of/CD18LZ or LFA-1LZ, and at 4 ℃ of incubation 6-8 hours.Plate then uses the TBS (not containing Tween_) of the every kind of calcium chloride, magnesium chloride and the manganese chloride that contain 2mM to wash once with Tris buffer salt solution (TBS-T) washed twice that contains Tween_20.The latter is used as test and lavation buffer solution in remaining test.

After catching integrin, plate washs three times with the TBS in 250 μ l/ holes.The CAM/Ig (seeing embodiment 12) of purification is added every hole through 2: 3 dilution backs of series from initial concentration 10 to 20 μ g/ml.With CAM/Igs after room temperature combines 2 hours, plate washs by preceding method.Bonded fusion rotein detects with the goat-anti people Ig antibody (Jackson Labs) of horseradish peroxidase, and then develops the color with o-phenylenediamine (OPD).

The result shows, when ICAM-1/Ig and LFA-1LZ coupling, 5 to 7 times of signal increases, but not with α _d/ CD18LZ coupling.On the contrary, VCAM-1/Ig is containing α _dShow the signal higher 5 times in the hole of/CD18LZ than background.A kind of ICAM-R mutant E37A/Ig (seeing embodiment 12) not with two kinds of integrin couplings.

Detected α _dMonoclonal antibody specific 212D, 217L, 217I, 217H, 217G, 217K and 217M suppress VCAM-1 and immobilized α _dThe link coupled ability of/CD18.In addition, use anti-VCAM-1 monoclonal antibody 130K, 130P and IG11B1 (Caltag) to determine the specificity of reaction.Anti-α _dThe working concentration of monoclonal antibody is 5 μ g/ml, and the working concentration of anti-VCAM-1 antibody is 25 μ g/ml, considers that the VCAM-1 in this pilot system is in the solution, therefore uses higher anti-VCAM-1 antibody concentration.

In the hole that 130P handles, producing partly blocking-up (50%) with 217I or 130K.The combination of 130K/130P has also suppressed reacting to each other of VLA-4 and VCAM-1 fully, and this illustrates α _dCombine with the different loci of VCAM-1 with VLA-4, and might develop and optionally intervene α _dThe bonded antagonist of/VCAM-1.

This test can be carried out high-throughout Screening test as described below, screening α _dBonded mortifier.The VCAM-1/Ig biotinylation is also used when having the mixing cpd that is dissolved in advance among the DMSO as stated above, use a kind of streptavidin-europium (Eu) complex to detect bonded VCAM-1/Ig then.Be activated the light emission that generation can be measured behind streptavidin-Eu complex chelating.Photoemissive change, or reduce in particular, VCAM-1/ α just shown _dBonded inhibition, this is likely the result of one or more compound effects in the small-molecule mixture, just can carry out separate analysis or be divided into little group analyzing to them then.

Embodiment 14

Soluble human α _dExpression construct

Total length, soluble people α _dThe proteic expression of/CD18 heterodimer can be easily for immunity with in conjunction with measuring the material that purification is provided.The advantage of preparation soluble protein be its can be from supernatant rather than from cell lysate purification (as with the film of total length in conjunction with α _d/ CD18), at this moment, yield improves and impurity reduces.

Solubility α _dExpression plasmid is performed as follows structure.Be cloned in nucleotide fragments among the plasmid pATM.D12, that be equivalent to the 0-3161 base zone among the SEQ ID NO:1, separate with the method for AatII digestion with HindIII.It is the base 3130-3390 that is equivalent to SEQID NO:1, overlapping and contain the PCR fragment of an extra MluI restriction site at 3 ' end with the HindIII/AatII fragment to use the primer sHAD.5 list in SEQ ID NO:30 and SEQ IDNO:31 respectively and sHAD.3 to increase one from plasmid pATM.D12.

5′-TTGCTGACTGCCTGCAGTTC-3′ (SEQ?ID?NO：30)

5′-GTTCTGACGCGTAATGGCATTGTAGACCTCGTCTTC-3′

(SEQ?ID?NO：31)

The PCR product digests with AatII and MluI, and is connected with the HindIII/AatII fragment.The product that is obtained is connected among the plasmid pDC1.s of HindIII/MluI digestion.

The coexpression in the Chinese hamster ovary celI of stable transfection with this construct and CD18, express use from ³⁵The radioactive automatic developing of the CD18 complex of immunoprecipitation detects in the cell of S-methionine labelling.Coexpression [Berman, et al., J.Cell.Biochem.52:183-195 (1993)] in 293 cells also simultaneously with this construct and CD18.

Solubility total length α _dConstruct

The present invention also comprises other α _dExpression construct is for ease of expressing and a kind of complete α of purification _d/ CD18 heterodimer, solubility α _dBe built as with the CD18 expression plasmid and comprise that a kind of " leucine zipper " fusion sequence, this sequence make heterodimer stable [Chang, etal., Proc.Natl.Acad.Sci. (USA), 91:11408-11412 (1994)] in purge process.In brief, the DNA of coding acidity of slide fastener and basic amino acid chain carries out primer annealing with the oligonucleotide of introductions such as Chang and prepares.This DNA sequence further modified respectively 5 ' and 3 ' end comprise extra MluI and XbaI restriction site, make this DNA sequence be convenient to sub-clone to foregoing α _dOr in the CD18 expression construct.In addition, add sequence and termination codon of representing a hemagglutinin or a polyhistidyl sequence in back, XbaI site.Integration hemagglutinin or polyhistidyl are the purification for the ease of expressing protein.The sequence of the alkaline chain of coding slide fastener is incorporated on the plasmid vector of expressing CD18, and acid chain is inserted into α _dOn the chain structure.The α that modifies _dWhen in host cell, expressing, infer that the acid chain of zipper and the interaction between the alkaline chain will make heterodimer stable, and make complete α with CD18 albumen _dThe enough affinity purifications of introducing above of/CD18 molecular energy separate.

Structure has the α of the expression solubility of acid and alkalescence " leucine zipper " sequence _dWith the plasmid of CD18, according to the DEAE/Dextran method rotaring redyeing COS cell of embodiment 7 introductions.The albumen that is obtained is called as " α _d/ CD18LZ ".Hemagglutinin and polyhistidine tag are not incorporated into α _dAmong/the CD18LZ.Transfectional cell was grown 14 days under reduction serum (2%) condition.From transfectional cell, collect supernatant with the ELISA method of embodiment 8 introductions every 5 days and detect proteic generation.In brief, with α _d/ CD18LZ is immobilized in and uses anti-α _dOn the plate of monoclonal antibody 169B (seeing embodiment 15) bag quilt.Add biotinylation anti-CD18 monoclonal antibody TS1/18.1 (seeing embodiment 8), then add streptavidin/horseradish peroxidase (HRP) conjugate and o-phenylenediamine (OPD), detect α _d/ CD18LZ complex.Albumen in the supernatant can clearly detect.

Use solubility total length α _dThe combination of expression product is measured

The solubility total length α that use is introduced above _dThe functional combination of/CD18LZ heterodimer is measured by carrying out on the plate that heterodimer is immobilized in monoclonal antibody 169B or the anti-CD18 monoclonal antibody of a kind of non-barrier (seeing embodiment 15) bag quilt.Hole (seeing embodiment 12) before adding the CAM/Ig chimera that initial concentration is 10 μ g/ml is sealed to prevent non-specific binding with fishskin gelatin.Chimera and α _dThe combination of/CD18 detects with goat-anti people Ig HRP conjugate (Jackson Labs), and then develops the color with OPD.

The α that VCAM-1/Ig is observed and catches _dThe binding ratio of/CD18LZ and the high 3-5 of the CD11a/CD18 that catches are doubly.High about 15 and 10 times respectively of the binding ratio backgrounds of the CD11a/CD18 heterodimer of ICAM-1/Ig and ICAM-2/Ig and solubility, but not with α _d/ CD18 combination.Being combined in when having VCAM-1 specific antibody 130K and 130P coupling of VCAM-1 lowers about 50%.

Use is immobilized in the ICAM/Ig albumen on 96 orifice plates and adds recombinant soluble integrin in the cell conditioned medium liquid subsequently also have been carried out in conjunction with measuring.The combination of solubility integrin is followed and the link coupled sheep anti-mouse antibody incubation of HRP with cold non-barrier α and the special murine antibody of β subunit, and with the OPD colour developing, detects.

The result shows, the detected α of non-blocking antibody _d/ CD18LZ combines with ICAM-R/Ig's, and is than detected combination in the control wells that does not contain antibody, high 10 times.Do not detect solubility α _d/ CD18 combines with immobilized ICAM-1/Ig's, but at α _dThe observed combination between/CD18 and immobilized CD11b/CD18 and the CD11a/CD18, respectively than background in conjunction with high 15 and 5 times.

Because research in the past is verified, CD11b and CD11c combine [Wright, Curr.Opin.Immunol.3:83-90 (1991) with lipopolysaccharide (LPS); Ingalls andGolenbock, J.Exp.Med.181:1473-1479 (1995)], LPS and α _dThe combination of/CD18 is also estimated with flow cytometry and plate based assays.The result shows, can be faintly when the 20 μ g/ml and α from the LPS (all available from Sigma) of salmonella minnesota and the isolating FITC labelling of salmonella typhi _dThe Chinese hamster ovary celI combination of/CD18 transfection.In the contrast Chinese hamster ovary celI of untransfected, do not find combination.In ELISA type test, biotinylated LPS[Luk, et al., Alan.Biochem.232:217-224 (1995)] when 0.5-3.0 μ g in conjunction with the α that is obtained _d/ CD18LZ signal is higher 4 times than independent capture antibody and blocking-up reagent.LPS and CD11a/CD18 apparent is combined in to have got rid of and combines the back with anti-CD11a antibody TS2/4 at the bottom of each advance copy and reduce.

For identifying α _dOther parts of/CD18 have used recombinant alpha in one two level research _d/ CD18LZ albumen.Different cell types are used for determining with proteic combination of immobilization which kind of cell is at cell surface expression α _dPart.Suppress to determine that with antibody viewed cell is in conjunction with whether known surface adhesion molecule causes then.If do not suppress the result, use α _d/ CD18LZ with come self energy and α _dThe proteic co-immunoprecipitation of the lysate of bonded cell manages to identify part.

Soluble human α _dI domain expression construct

There has been the I domain among the report CD11a to express, and can have kept its ligand binding capacity and antibody recognition characteristic [Randi and Hogg, J.Biol.Chem.269:12395-12398 (1994) as an independent structures unit; Zhout, et al., J.Biol.Chem.269:17075-17079 (1994); Michishita, et al., Cell 72:857867 (1993)].For preparing a kind of α of comprising _dThe soluble fusion protein of I domain and human IgG 4 is used pcr amplification α _dThe I domain uses design to add BamHI and XhoI restriction site so that the primer of sub-clone at flank.Primer is listed in SEQ ID NO:32 and 33, and at the restriction enzyme site underscore.

5′-ACGTATGCA GGATCCCATCAAGAGATGGACATCGCT-3′

(SEQ?ID?NO：32)

5′-ACTGCATGT CTCGAGGCTGAAGCCTTCTTGGGACATC-3′

(SEQ?ID?NO：33)

Among the SEQ ID NO:32 and then the C nucleotide of BamHI site 3 ' end be equivalent to 435 nucleotide of SEQ ID NO:1, among the SEQ IN NO:33 and then the G nucleotide of XhoI site 3 ' end be complementary to 1067 nucleotide among the SEQ ID NO:1.The I domain of amplification is with suitable enzymic digestion, the fragment of purification is connected to mammalian expression vector pDCs and prokaryotic expression carrier pGEX-4T-3 (Pharmacia), and I domain sequence is checked order.Use COS, CHO or the Bacillus coli cells expressed fusion protein of suitable expression construct transfection or conversion then.

Known α _dICAM-R had affinity, α _dThe expression of I domain may have enough affinitys and come as α _dThe inhibitor of the cell adhesion that participates in.

People α _dThe analysis of I domain/IgG4 fusion rotein

Protein is used in that SDS-PAGE analyzes under reduction and the non-reduced condition, and dyes or coomassie dyes and to show with silver.Then albumen is transferred on the Immobilon pvdf membrane, and carried out the Western engram analysis with anti-human IgG monoclonal antibody or anti-cattle Ig monoclonal antibody.

Detected albumen is determined under the non-reduced condition and moves with about 120kD, moves with 45kD under reducing condition.Under non-reduced condition, also detect less important band, it can be with anti-people's antibody response but not with anti-cattle antibody response.The less important band of a 200kD is defined as cattle Ig by the Western trace.

Use the combination of I domain expression product to measure

In an ELISA test, detected the ability of I domain specific recognition ICAM-R/IgG chimeric protein.The α of serial dilution in TBS _dI domain IgG4 fusion rotein (I α _d/ IgG4) with the ICAM-1/IgG that is immobilized onto Immulon_IV RIA/EIA plate, ICAM-R/IgG, VCAM-1/IgG or a kind of irrelevant IgG1 myeloma protein incubation.CD11a I domain/IgG chimeric protein and human IgG 4/ κ myeloma protein are as negative control.Bonded IgG4 is with biotinylated anti-IgG4 monoclonal antibody HP6023, and then adds streptavidin-horseradish peroxidase thing, with the colour developing of substrate o-phenylenediamine, detects.

In repeated in experiments, do not detect the combination of CD11a/IgG4 albumen or IgG4 myeloma protein with any immobilization albumen.I α _d/ CIgG4 albumen does not combine with fishskin gelatin or bovine serum albumin closed reagent, human IgG1 or ICAM-1/IgG.Use the I α of 1-5 μ g/ml concentration _d/ IgG4 albumen has detected the binding signal doubly than the high 2-3 of background in the hole of ICAM-R/IgG albumen bag quilt.Signal in the hole of VCAM-1/IgG albumen bag quilt than the high 7-10 of background doubly.In the test in front, α _dThe Chinese hamster ovary celI of/CD18 transfection can not be in conjunction with VCAM-1/IgG albumen, and this shows that the VCAM-1 combination may be the feature of isolating I domain aminoacid sequence.Other α _dI domain construct

Other α _dI domain construct uses the mode identical with the construct of front to prepare, but around α _dThe I domain is integrated more aminoacid.Concrete construct comprises: i) from exon 5 (the 127-353 aminoacid of SEQ ID NO:2), in the front of current construct; Ii) the EF chirality after the I domain repeats (the 17-603 aminoacid of SEQ ID NO:2); Iii), have an IgG4 tail that is used for purification and detection at the α chain (the 17-1029 aminoacid of SEQ ID NO:2) that changes the diaphragm area truncate.These constructs are connected among mammalian expression vector pDCS1 or the prokaryotic expression carrier pGEX-4T-3 (Pharmacia), and the I domain is checked order.Then transform with suitable expression construct or COS, the CHO of transfection or Bacillus coli cells in expressed fusion protein.(England) post carries out purification to albumen for Bioprocessing Limited, Durham, and with anti-IgG4 monoclonal antibody HP6023 detection reaction, shows with coomassie dyeing on polyacrylamide gel with ProSepA_.

The α that is used for The expressed for structure _dThe polypeptide expression plasmid is modified the pATM.D12 that above introduces by following method and to be expressed a kind of α _d-IgG4 fusion rotein.The DNA of coding IgG4 separates from carrier pDCS1 with the method for PCR, uses the primer of having integrated 5 ' AatII restriction site (SEQ ID NO:89) and 3 ' XbaI restriction site (SEQ ID NO:90) respectively.

5′-CGCTGTGACGTCAGAGTTGAGTCCAAATATGG-3′ (SEQ?ID?NO：89)

5 '-GGTGACACTATAGAATAGGGC-3 ' (SEQ ID NO:90) plasmid pATM.D12 is with AatII and XbaI digestion, and the IgG4 PCR product that will suitably digest with purification is connected in the carrier of a linearity.

Embodiment 15

Preparation people α _dSpecial monoclonal antibody

1. will in Dulbecco ' s phosphate-buffered saline (D-PBS), wash three times from the transient transfection cell of embodiment 7, by 5 * 10 ⁶Cell/Mus is expelled in the Balb/c mice with the muramyldipeptide (Sigma) of 50 μ g/ Mus.Mice was injected twice at interval with same mode in 2 weeks again.Before the blood-letting of mice and immune serum screens with the method for embodiment 9 introductions, will with express alpha _dThe transfectional cell of/CD18 has the spleen of the mice of highest response to merge.Screen cell and the screening and the α of the COS cell effect of shortage and CD11a/CD18 transfection then respectively _dThe hybridoma supernatant of the cell effect of expression plasmid and CD18 cotransfection.

Do not obtain monoclonal antibody with the method.

2. another kind of preparation monoclonal antibody method is, the α of affinity purification solubility from the supernatant of the Chinese hamster ovary celI of stable transfection _dI domain/IgG4 fusion rotein, and immune as stated above Balb/c mice.Set up hybridoma, and screen energy and α with ELISA _dThe supernatant from these hybridomas of I domain fusion rotein reaction.Analyze the total length α that expresses on positive culture and the CHO transfectional cell then _dThe reactivity of/CD18 complex.

Mice 1908 is accepted three α _dThe initial immunity of the Chinese hamster ovary celI of/CD18 transfection and twice solubility α _dThe booster immunization of/CD18 heterodimer.Last twice immunity comprises the α of 50 μ g/ Mus _dI domain/IgG4 fusion rotein.Merge and produced 270 holes that produce IgG.The supernatant in 45 holes shows in ELISA and I α _dThe binding ratio of/IgG4 fusion rotein is high at least 7 times with combining of human IgG 4.Facs analysis determines do not have supernatant energy and α _dThe Chinese hamster ovary celI reaction of/CD18 transfection.

For determining whether supernatant can discern beta 2 integrin alpha subunit under another kind of environment, fresh freezing spleen section is dyeed with 24 hole supernatant in 45 holes, and three supernatant are confirmed as the positive.Caught a large amount of cells in the red pulp for one, and two other has caught the cell dispersion in red pulp and the girder.

These supernatant immunoprecipitations have further been analyzed from α _dThe ability of the biotinylation CD18 of the HL60 cell that the Chinese hamster ovary celI of/CD18 transfection or PMA stimulate.Further sub-clone is carried out in the fusion hole of selecting its supernatant can discern the albumen (being not limited to the albumen with the heterodimer formal representation on this protein structure) in the detergent lysate.Can discern proteic monoclonal antibody in the detergent may be more useful during from the heterodimer complex of transfectional cell, tissue and cell line at immunoprecipitation.

3. the alternative method of another kind of preparation monoclonal antibody is that the CD18 complex is being removed CD11a/CD18 (using monoclonal antibody TS2/4) and CD11b/CD18 (using monoclonal antibody Mo-1) back immunoprecipitation from people's spleen lysate in advance with anti-CD18 monoclonal antibody 23F2G.The Balb/c mice in 5 10-12 age in week carried out immunity with hypodermic method with the albumen that obtains of about 30 μ g in Freund's complete adjuvant at the 0th day, then carried out booster immunization at the 28th and the 43rd day twice by 30 μ g/ Mus and with incomplete Freund.Extracted test serum the last time behind the booster immunization in 10 days.Every kind of serum with dilution in 1: 500 in the Western trace detects the immunogenic method of 1 μ g/ bar reactivity is estimated.Can detect about 95 and the band of 150kD from the serum of three mices, in the bar of handling with the preimmune serum of dilution in 1: 50, not find signal.Infer that 150kD represents the α of glycosylation state in a kind of body _dIn addition, all through the serum of immunity can both immunoprecipitation from biotinylation α _dThe albumen of the lysate of/CD18 Chinese hamster ovary celI, this albumen move with suitable molecular weight on SDS-PAGE, and represent heterodimer.By these results, select mice #2212,64 days with 30 μ g immunogens among the PBS, use the method for peritoneal injection, further immunity.After 4 days, put to death mice, aseptic taking-up spleen.

The frosted end of two microscope slides of spleen in being immersed in the serum-free RPMI 1640 that adds 2mM L-glutamic acid, 1mM Sodium Pyruvate, 100 units/ml penicillin and 100 μ g/ml streptomycins is ground, the suspension (RPMI) of formation individual cells (Gibco, Canada).Cell suspension filters 70 aseptic sieve Nitex cell filters (Becton, Dickinson, Parsippany, New Jersey).Permeate is 200 * g washing in centrifugal 5 minutes 2 times.The precipitation that is obtained is suspended in 20ml serum-free RPMI again.The thymocyte cell that takes out from three Balb/c children Mus also uses the same method and is prepared.

Before fusion, will (RPMI Utah) maintains the NS-1 myeloma cell of exponential phase for Hyclone Laboratories, Logan, precipitates in centrifugal 5 minutes at 200 * g with containing 10%Fetalclone serum (FCS) merging first three day.With the method washed twice of the preceding paragraph introduction, and counting.With about 2 * 10 ⁸Splenocyte and 4 * 10 ⁷The NS-1 mixing with cells, with the mixture that obtained with 200 * g centrifugation.Supernatant discards.Knock test tube and take out cell precipitation, and in 1 minute, add 50%PEG1500 among the 2ml 75mM Hepes (pH8.0,37 ℃) (Boehringer Mannheim), and stir.In 7 minutes of following, add other 14ml serum-free RPMI, then add the RPMI of 16ml immediately.The mixture that is obtained centrifugal 10 minutes at 200 * g, abandoning supernatant.The precipitation be suspended in again 200ml contain 15%FBS, 100mM hypoxanthine sodium, 0.4mM aminopterin, 16mM thymus pyrimidine (HAT) (Gibco), 25 units/ml IL-6 (Boehringer Mannheim) and 1.5 * 10 ⁶The RPMI of thymocyte cell/ml, and divide in the flat tissue culturing plate in 96 holes (Corning, the United Kingdom) by 200 μ l/ holes.The the 2nd, the 4 and 6 day cell of feeding after fusion, method is with the about 100 μ l of a 18G pin (Becton Dickinson) sucking-off from every hole, and add the bed board culture medium introduce above, but contain the IL-6 of 10 units/ml and do not contain outside the thymocyte cell by 100 μ l/ holes.

Screen the supernatant in every hole with antibody capture ELISA after fusion 7-10 days, detects the existence of Mus IgG.(Massachusetts) the sheep anti mouse IgA, the IgG that are diluted in 50mM sodium carbonate buffer, pH9.6 at 1: 5000 or the IgM (Organon Teknika) with 50 μ l/ holes wraps quilt at 4 ℃ to the Immulon_IV plate for Dynatech, Cambridge.Plate washs three times with the PBS (PBST) that contains 0.5%Tween_20, and adds the culture supernatant 50 μ l from each hole.After 30 minutes, wash as stated above with PBST in the hole at 37 ℃ of incubations.In every hole, add then 50 μ l by the sheep anti-mouse igg that is diluted in the horseradish peroxidase of PBST at 1: 3500 (Fc) (Jackson ImmunoRsesearch, WestGrove, Pennsylvania).Plate carries out incubation as stated above, with PBST washing four times, is added in then and contains 1mg/ml o-phenylenediamine (Sigma) and 0.1 μ l/ml 30%H among 100mM citric acid, the pH4.5 ₂O ₂Substrate.The sulphuric acid that added 50 μ l 15% after 5 minutes stops color reaction.The 490nm that determines every hole with plate reading machine (Dynatech) absorbs.

Hybridoma is further identified in the following method.From the supernatant that produces the IgG culture with flow cytometry itself and α _dThe Chinese hamster ovary celI that/CD18 transforms rather than (find a kind of LFA-1 positive, but other β in the original position dye test in front with the JY cell ₂The B cell line of integrin feminine gender) reactivity.In brief, with 5 * 10 ⁵α _dChinese hamster ovary celI or α that/CD18 transforms _d/ CD18 ^-The JY cell suspension contains 2%FBS and 10mM NaN in 50 μ l ₃Among the RPMI of (FACS buffer), respectively each cell suspension is added in the 50 μ l IgG positive hybridoma culture supernatants in the 96 hole circle base plates (Corning), at incubation on ice after 30 minutes, cell is with twice of medical centrifuge washing of precipitate.The supernatant in every hole is discarded, and will precipitate and be suspended in 200-300 μ l FACS buffer again.The last F (ab ') that washs with the sheep anti-mouse igg that in the FACS buffer, prepares (the H+L)-FITC conjugate that dilutes at 1: 100 in 50 μ l/ holes ₂Replace.Behind the incubation, cell is with having added 10mM NaN as stated above ₃Dulbecco ' s PBS (D-PBS) washed twice.And be suspended in the D-PBS that contains 1% paraformaldehyde at last.Then with sample transfer in the polypropylene test tube, carry out flow cytometry (FACs) with BectonDickinson FACsan analyser.

Merge and produced four by two kinds of seemingly male cultures of standards judgement.When after about 4 days, carrying out repeating to screen second time, there are three to keep the positives in four cultures with the supernatant that enlarges.These three hole called after 169A, 169B, 169D clone these three holes 2 to 3 times continuously by the dilution of twice in the RPMI of 15%FBS, 100mM hypoxanthine sodium, 16mM thymus pyrimidine and 10 units/ml IL-6.The range estimation counting writes down the clone's number in the minimum density hole after Kong Zaisi days on clone's plate.Each clone's selecting hole was analyzed with FACS after 7-10 days.In two culture 169A and 169B, find active.Among the clone, the positive hole of containing single clone enlarges in containing the RPMI of 11%FBS the last time.Antibody in 169A and the 169B clone supernatant carries out typing with IsoStrip test kit (Boehringer Mannheim) according to manufacturer's indication, finds that they belong to the IgG1 isotype.

α with the HL-60 cell that stimulates from CHO transfectional cell and PMA _dThe immunoprecipitation of/CD18 complex is used as specific four-wheel screening.Hybridoma 169A and 169B can precipitate from the suitable band of Chinese hamster ovary celI system and from a single α chain type of HL-60 cell, and this result is determined by SDS-PAGE.Hybridoma 169A and 169B May 31 nineteen ninety-five in American type culture collection, 12301 Parklawn Drive, Rockville, Maryland 20852 has carried out preservation, its preserving number is respectively HB11907 and HB11906.

For analyzing 169A and 169B binding characteristic more completely, detected every kind of antibody and suppressed another kind of antibody or anti-CD18 antibody TS1/18.1 and solubility α _dThe bonded ability of/CD18.The α of solubility total length _d/ CD18 is detected identical or different non-labeled antibody with biotinylated antibody and combines with proteic by every kind in 96 orifice plates unlabelled antibody immobilization respectively.Use a kind of sheep anti mouse Ig/HRP conjugate and then add the OPD substrate and detect combination.The result shows that antibody 169A can block the combination of biotinylation 169A and TS1/18.1, and antibody 169B only blocks the combination of himself.

4. select another mice (#2214), use the method identical to carry out initial immunity with mice #2212, and the booster immunization before 70 days merge, the 30 μ g purification α from the spleen lysate among the PBS are used in immunity _dPut to death mice after 4 days, aseptic taking-up spleen.The fusion of positive cell and clone carry out as mentioned above.Merge and produced 5 anti-α _dThe monoclonal hybridoma, called after 170D, 170F, 170E, 170X and 170H use IsoStrip test kit (Boehringer Mannheim) to carry out typing according to manufacturer's indication, find that they belong to the IgG1 isotype.

5. select another mice (#2211), use the method identical with mice #2214 to carry out initial immunity, carried out booster immunization with 30 μ g immunogens, the booster immunization before 203 days merge with 30 μ g immunogens at 88 days with mice #2212.Mice was put to death after 4 days, took out spleen and fusion according to aforesaid method.Hybridoma is according to the detailed introduction of top paragraph, by antibody capture ELISA and flow cytometry screening hybridoma supernatant.

Identified 15 male hybridomas, called after 188A, 188B, 188C, 188E, 188F, 188G, 188I, 188J, 188K, 188L, 188M, 188N, 188P, 188R and 188T, and carry out typing with ELISA.In brief, the Immulon_4 plate (Dynatech, Cambridge, Massachusetts) by 50 μ l/ holes be used in 50mM sodium carbonate buffer, pH9.6 by sheep anti mouse IgA, the G of dilution in 1: 5000, M (Organ Teknika) at 4 ℃ of bag quilts.Plate 37 ℃ of sealings 30 minutes, with PBS/0.5%Tween_20 (PBST) washing three times, adds the culture supernatants (use PBST1: 10 dilute) of 50 μ l with the PBS that contains 1%BSA.After carrying out incubation and washing as previously mentioned, add 50 μ l with the PBST that contains 1% normal sheep serum by rabbit anti-mouse igg 1, G2a or the G3 of the horseradish peroxidase of dilution in 1: 1000 (Zymed, SanFrancisco, California).Plate with PBST washing four times, adds 100 μ l substrates by the aforementioned incubation that carries out then, and substrate is for containing 1mg/ml o-phenylenediamine (Sigma) and 0.1 μ l/ml 30%H ₂O ₂100mM citric acid, pH4.5.The sulphuric acid that added 50 μ l 15% after 5 minutes stops color reaction.(Dynatech) reads A with plate reading machine _490nmAll 15 antibody are confirmed as IgG1.

Will be freezing in a freezing tubule from the residue splenocyte of mice #2211, and be stored in liquid nitrogen.Freezing tubule placed in 37 ℃ of water baths thaw rapidly, and it is moved in the circumference mode, melt until content.Cell is gone to a 15ml centrifuge tube, slowly add the preheating RPMI that 1ml contains 11%FBS simultaneously, adition process is 3 to 5 minutes.Wait for after 5 minutes, add the RPMI of 5ml preheating again, test tube centrifugal 5 minutes, sucking-off supernatant at 200 * g.Cell is suspended in RPMI again, merges by preceding method.The hybridoma supernatant is screened with antibody capture method and flow cytometry by preceding method.

Merge and produced 5 clones, called after 195A, 195C, 195D, 195E and 195H.By preceding method these clones are carried out typing with the ELISA program, monoclonal antibody 195A, 195C, 195D and 195E are confirmed as IgG1, and 195H is confirmed as IgG _2a

6. at the anti-α of preparation _dIn another effort of monoclonal antibody, mice #2213 uses with the same method of mice 2214,2211 and 2212 and carries out immunity, but uses and the link coupled 30 μ g people α of Sepharose_ pearl at the 414th day and 441 days _d/ CD18 leucine zipper (LZ) carries out further immunity.The immunogen that is used for mice #2213 is passed through people α _d/ CD18LZ (embodiment 14) prepares with anti-CD18 monoclonal antibody and protein A Sepharose_ immunoprecipitation.Sedimentary complex is resuspended in PBS in 1: 1 ratio becomes slurry.4 days execution mices behind booster immunization.Take out spleen, merge by preceding method.

Male hybridoma is identified with the ELISA method, uses the F (ab) ' with the anti-CD18 antibody of non-barrier ₂The immobilized people α of fragment _d/ CD18LZ.In brief, F (ab) ' ₂Fragment is pressed 100ng/ hole bag quilt in the Immulon_4 elisa plate at 4 ℃.After the sucking-off buffer, the hole was sealed 30 minutes at 37 ℃ with 0.5% fishskin gelatin (Sigma).After in PBST, washing three times, add 50 μ l/ holes from the previous coding solubility α that uses _dThe supernatant of the Chinese hamster ovary celI that the plasmid of/CD18LZ transforms.Plate was 37 ℃ of incubations 30 minutes.The repeated washing step, and add the hybridoma supernatant in 50 μ l/ holes.The detection of monoclonal antibody is undertaken by preceding method.Positive cell is analyzed with flow cytometry by preceding method, uses α _dThe Chinese hamster ovary celI that/CD18LZ coding DNA transforms, two positive hybridomas that are named as 212A and 212D obtain identifying.The excretory antibody of hybridoma uses the typing ELISA typing of introducing previously to be IgG1.

7. prepare anti-people α at another _dIn the monoclonal antibody method.Mice is with pressing the α that preceding method prepares _d/ CD18LZ Sepharose_ pearl carries out immunity, and every mice was at the 0th day, 36 days and accepted the immunogen of 30 μ g in 66 days.After mice serum being screened, select mice #2477 to merge with the recombiant protein ELISA program of introducing previously.The method of introducing in 212 fusions above fusion, selection and clone's program are used is carried out.7 positive hybridomas---217F, 217G, 217H, 217I, 217K, 217L and 217M have been identified, but in the end one have taken turns among the clone hybridoma and lost reactivity that this point is determined by flow cytometry.Antibody from 6 hybridoma systems of residue carries out typing by preceding method, all is found to be IgG1.

8. at another preparation α _dIn the monoclonal antibody method, mice #2480 carries out immunity with the same method of mice #2477, but at the 217th day and 218 days α with 30 μ g _d/ CD18LZ carries out further immunity through peritoneal injection.Mice was put to death at the 221st day, took out spleen, and merged by preceding method.The hybridoma supernatant screens by the ELISA method of introducing, and determines it and before used coding for alpha with flow cytometry _dThe reactivity of the JY cell of the DNA transfection of/CD18.Screening process is undertaken by top introduction.Fusion has produced 3 male hybridomas, 240F, 240G and 240H, and three kinds of excretory antibody of hybridoma are IgG1 with the equal typing of ELISA method.

9. can inhibit feature α for identifying _dLink coupled antibody uses solubility α _d/ CD18LZ (seeing embodiment 14) carries out immunity.Albumen separates from the supernatant of the COS cell of transient transfection on a kind of affinity chromatograph resin, with the α of resin-bonded _dAs immunogen.A selected mice carries out immunity by the method for introducing above, and two weeks gives last reinforcement behind initial immunity.Can prevent may changing of often relevant protein conformation with this technology immunity with the detergent cracking of cell.Other mice is with also carrying out immunity with the recombiant protein of resin-bonded, but need not carry out initial immunity by the albumen of purification from cell lysate.

Press hybridoma preceding method preparation, that produce by immunity with ELISA at Fab fragment enterprising row filter of immobilized recombiant protein from cell conditioned medium liquid with a kind of non-blocking antibody.In addition, use flow cytometry its with before used α _dThe reactivity of the JY cell of cDNA transfection.

10. another kind of selecting method, monoclonal antibody is performed as follows preparation.Use is from the α of the affinity purification of the detergent lysate of the Chinese hamster ovary celI of stable transfection _d/ CD18 and 50 μ g/ml muramyldipeptides are pressed preceding method immunity Balb/c mice.By determining that with the biotinylation complex immunoprecipitation in the CHO transfectional cell it is to α _dBefore the serum reactivity of/CD18, mice is accepted three immunity.The hybridoma of positive animal is set up by standardization program.Use α then _d/ CD18 transfectional cell is selected the hybridoma culture with flow cytometry.The CD11a/CD18 transfectional cell as only with the contrast of CD18 reaction.

11. the selecting method of another kind of Monoclonal Antibody is, the Balb/c mice is accepted a kind of immunity/immunosuppressant program, this immunity/immunosuppressant program be designed to reduce with immunity in the reaction of Chinese hamster ovary celI determiner on the transfectional cell that uses.This program comprises that the Chinese hamster ovary celI with untransfected carries out immunity, and then handles the B cell precursor that kills with the Chinese hamster ovary celI reaction with cyclophosphamide.After immunity and cyclophosphamide processing through three-wheel, mice is by preceding method α _d/ CD18 CHO transfectional cell carries out immunity.

12. another kind of selecting method is, by the CD18 complex of aforesaid pre cleaning method enrichment from the detergent lysate of the HL60 cell of PMA stimulation.Other β ₂Integrin is eliminated on same post.Carrying out immunity, hybridoma preparation and screening process with the complex that is obtained is all undertaken by method mentioned above.

The preparation of polyclonal serum

Use the α of purification _dI domain/IgG4 chimera (embodiment 14) prepares polyclonal antiserum in rabbit.With the α in the Freund's complete adjuvant _dI domain/IgG4 antigen carries out initial immunity by 100 μ g/ rabbits, and then the albumen with the equal number in the incomplete Freund carries out booster immunization three times.At third and fourth injection back test blood to be measured.Rabbit immunoglobulin (Ig) on a protein A-Sepharose_ post from serum purification, and on a people IgG/Affigel_10 post the anti-human IgG reactivity of pre cleaning.Being used among the ELISA with the reaction of I domain chimera but not reacting with human IgG proves completely and removes.

The polyclonal serum of pre cleaning is used to from before using α _dWith immunoprecipitation albumen in the detergent lysate of the Chinese hamster ovary celI of the surface biological elementization of CD18 expression vector transfection.Immunoprecipitation is undertaken by the method for introducing among the front embodiment 10.The serum of pre cleaning is discerned a kind of albumen composition, and this albumen composition is with identical with the sedimentary molecular weight of albumen of anti-CD18 monoclonal antibody TS1.18.In addition, serum with from α _dWhen the CD18 complex of the Chinese hamster ovary celI of/CD18 transfection carries out the Western trace, discern a suitably single band of size.Integrin CD11a/CD18, CD11b/CD18 and VLA4 by people's spleen affinity purification are not discerned by rabbit polyclonal serum.Flow cytometry measure to find, this serum can not with the α in the solution _dThe Chinese hamster ovary celI reaction of transfection.Therefore conclusion is that rabbit polyclonal serum only can be discerned the α of degeneration _dI domain/IgG4 albumen.

At an anti-α of preparation _dIn the effort of/CD18 polyclonal antiserum, a mice is used α _dThe Chinese hamster ovary celI of transfection (D6.CHO, α _d/ CD18) and auxiliary peptide immunity 3 times, with the α of purification _dThe immunity of/CD18 heterodimer once.Last booster immunization only comprises α _d/ CD18 heterodimer.About 100 μ l immune serums are by adding about 10 ⁸The Chinese hamster ovary celI of LFA-1 transfection was 4 ℃ of pre cleanings 2 hours.Detect the serum obtained when 1/5000,1/10000,1/20000 and 1/40000 dilution and the α on normal person's spleen _dReactivity.Polyclonal antibody has reactivity when 1/20000 dilution factor, and a little less than dyeing very in 1/40000 o'clock.

Embodiment 16

Use anti-α _dThe flow cytometry of monoclonal antibody

Using anti-α _dUse several former generations and immortalized cell line in the research that monoclonal antibody 212D, 217K and 217L carry out.Cell dyeing is undertaken by the method for introducing among the embodiment 7 and analyzes.The former generation CD8 that MAGE-3 (melanoma associated protein) peptide is special ⁺/ CD56 ^-And CD4 ^-/ CD8 ^-/ CD56 ⁺Primary cell line shows strong positive to CD11b and CD11c, but not by any α _dAntibody staining.MAGE-3 peptide specific cell uses lotus peptide antigen presenting cell (APCs, dendritic cell or mononuclear cell) to enlarge from the PERIPHERAL BLOOD MONONUCLEAR CELL group.In conjunction with Phenotypic Selection, produced the cloning cell lysis system of the target cell that to kill and wound the native protein that carries the peptide source specifically through repetitive stimulation under the restriction diluting condition.

From the dendritic cell of peripheral blood, when having cytokine IL-4 and GM-CSF, cultivate after 7 days, can be by antibody and the anti-α of 217L at CD11a, CD11b and CD11c _dAntibody dyes by force.In repeated in experiments, antibody 217D, 217K, 217I, 217H and 217M be not with these cells with from the reaction of the dendritic cell of different donors.After the 14th day of cultivation, the antigenic surface expression of 217L weakens, and dyeing was complete obiteration in the 21st day.In incubation, the expression of CD11b and CD11c remains on high level (than the background high 2-3 order of magnitude that dyes).

Embodiment 17

Person monocytic cell α _dExpression

Purification person monocytic cell from peripheral blood

Extract about 300ml blood from an aspiration donor and add (Sigma) 3.8% sodium citrate buffer solution.Blood is diluted to 480ml with no endotoxic PBS (Sigma), on the 17ml Histopaque of the careful layering of blood in the 50ml centrifuge tube with the 30ml dilution.In the BeckmanTabletop centrifuge, carried out layering in centrifugal 30 minutes with 1500rpm.Represent the cellular layer of mononuclearcell from every layer, to collect, and forward a new 50ml pipe to.With no endotoxic PBS, 0.1%BSA (no endotoxin) volume is increased to 50ml, in Beckman Tabletop centrifuge, use centrifugal 15 minutes of 1500rpm.Abandoning supernatant is resuspended in cell among the PBS/BSA of a small size, mixes subsequently.

Purification mononuclear cell layering for the second time from the mixing with cells group who obtains as stated above (use Percoll[Denholm and Wolber, J.Immunol.Meth.144:247-251 (1991)]).In brief, the Hanks buffer (Gibco) with 10ml 10X mixes with the 1N HCL of 600 μ 1.In this mixture, add the Percoll (Pharmacia, Piscataway NJ) of 60ml, make all Percoll enter solution slow stirring of mixture.The pH of Percoll solution is adjusted to 7.0, then the stepped mixing thing of 8.0ml is added the round bottom polypropylene test tube of 6 15ml.In each classification, accurately add 4ml cell suspension, and test tube put upside down it is fully mixed.Classification thing at room temperature centrifugal 25 minutes at 1690rpm with an angle rotor.Mononuclear cell level part is shown as the thin leucorrhea of one deck in the layering thing, with its collection and go in the new 50ml centrifuge tube.With PBS/0.1%BSA volume is adjusted to 50ml, centrifugation cell.Cell precipitation is suspended in the small size again, and mixes, measure cell quantity with hematimeter.Be resuspended in cell in the FACS buffer (RPI1640,0.2%FBS, 0.2% Hydrazoic acid,sodium salt) and be adjusted into 1.0 * 10 ⁶/ condition promptly uses 1.0 * 10 under every kind of FACS dyeing condition ⁶Cell is analyzed different cell markings.

FACS dyeing and analysis

Monoclonal antibody somatic cell dyeing is used α _dOr cell marking has immunologic opsonin and carries out with the direct link coupled antibody of a kind of label of available fluoroscopic examination.With mouse-anti people α _dAntibody 212D or 217L add cell by 10 μ g/ml, then incubation on ice 30 minutes, and wash 3 times.In other cell sample, add direct link coupled cell marking CD3-FITC of 10 microlitres (Becton-Dickinson) (T cell-specific) or CD33-FITC (Becton-Dickinson) (mononuclear cell is special), simultaneously that 10 μ l two are anti---anti-Mus FITC (Sigma) joins in 212D and the painted cell of 217L.All samples wash 3 times at incubation 30 minutes in the dark on ice, are resuspended in the paraformaldehyde of 300 μ 1 2%.With BectonDickinson FACScan on the sample, and with Lysys II software (Becton Dickinson) analytical data.

In first experiment, in all cells with two staging purification, mononuclear cell accounts for 68%, and the T cell accounts for 18%.There are a considerable amount of two kinds of cell types to carry out α at 212D and 217L _dCaught during dyeing, be respectively 55% and 65% of cell.Based on a back experiment, carry out α person monocytic cell to fresh separated _dAs if during dyeing, in the difference that exists on the relative populations between donor and the donor, though isolating mononuclear cell stained positive always.When in cell, adding human IgG (using in 10 minutes on ice with 1mg/ml before adding first antibody) when blocking any possible Fc receptors bind problem, α _dDyeing does not change.When these cells in the culture dish (Interferon Sciences) that uses Hydron bag quilt suspension culture and analyze α in 10%FBS/RPM-1640 _dDuring expression, forfeiture is just arranged, and should the minimizing process continue a time course of 7 days inner surface expression in 24 hours.Comprise CD11a, CD11b and the expression of CD11c on the person monocytic cell of fresh separated, α with respect to other integrins _dIt is lower to dye.

Person monocytic cell's α _d2 color FACS dyeing

For carrying out 2 color FACS dyeing, 212D and 217L antibody all use NHS-LC-biotin (Pierce) to carry out biotinylation according to the indication of manufacturer.In an independent experiment, cell separates as stated above, and dyes on ice 30 minutes with biotin labeled 212D and 217L antibody and a kind of biotin labeled contrast IgG1 antibody 10 μ g/ml.Cell washs three times in FACS buffer (be modified to and contain D-PBS, 2%FBS and 0.2% Hydrazoic acid,sodium salt), and is suspended in again in the FACS buffer of 1.0ml.10 link coupled CD33 of μ l FITC-(mononuclear cell is special) and 5 μ l streptavidin PE (PharMingen) are added in the cell suspension together.Incubation on ice 30 minutes, washing was three times in the FACS buffer, is resuspended in 300 μ l, 1% paraformaldehyde in the dark place for sample.Sample carries out FACS as stated above.

In two kinds of antibody, 217L show than contrast significantly with CD33 ⁺The cell combination.Antibody also can combine with this cell type, but painted CD33 ⁺The quantity of cell is starkly lower than observed quantity in antibody 217L.This result is consistent in two independent experiments.In the related experiment of using biotinylated antibody 212D and 217L, the 217L-biotin is always than the more cell of the biological uniformly dyeing of 212D-.

Represent the mononuclearcell of lymphocyte mixture and also detect with 2 colour analysiss of introducing above with the mononuclear cell that obtains before the Percoll gradient separations, it is link coupled to CD3 (T cell) to detect employing 212D and 217L-biotin and FITC, CD4 (helper T cell), CD5 (thymocyte cell, mature T cells, the B cell subsets), CD8 (cell toxicant/suppressor T cell), CD14 (mononuclear cell, neutrophilic granulocyte, the trifle dendritic reticulum cell), CD20 (B cell) and CD56 (NK cell, the T cell subsets) antibody of (Becton Dickinson) immunologic opsonin carries out two dyeing.The α that does not have discovery and these cell marking coexpressions _dPositive cell group.

Embodiment 18

α _dThe analysis that distributes

α _dThe tissue distribution of/CD18 uses the polyclonal antiserum of the method preparation of introducing by embodiment 15 to measure.

The concentration range that the rabbit polyclonal antibody of purification uses when immunocytochemical assay is carried out in section to refrigerated people's spleen as 120ng/ml to 60 μ g/ml.6 microns slabs are placed on Superfrost Plus slide (VWR) to be gone up and is stored in-70 ℃.Before the use, microscope slide is taken out from-70 ℃, placed 5 minutes at 55 ℃.To cut into slices then and in ice-cold acetone, fix 2 minutes, air drying.Section was being contained in the solution of 1%BSA, 30% normal human serum and 5% normal rabbit serum sealing 30 minutes in room temperature.First antibody is added each section room temperature treatment 1 hour.Unconjugated antibody is removed for three times by washed in the TBS buffer, washs 5 minutes at every turn.Then, will connect antibody at the rabbit anti-mouse igg in the same TBS buffer joins in each section.A kind of Mus alkali phosphatase alkali resistance phosphatase (APAAP) antibody room temperature incubation 30 minutes, is used for detecting two anti-.Then microscope slide is washed 3 times in the TBS buffer.Add Fast Blue substrate (Vector Labs), chromogenic reaction is stopped by immersing in the water.Microscope slide is negative staining in Nuclear Fast Red (Sigma), and rinsing in water, uses Aqua Mount (Baxter) counting then.In splenic red pulp, detect dyeing with this reagent, but do not detect dyeing with irrelevant rabbit polyclonal Ig prepared product or from the unpurified preimmune serum of same animal.

In case being determined, Mus serum has specific α _dActivity just comes different lymph samples or non-lymphoid tissue are dyeed with it.Identification CD18, CD11a, CD11b and CD11c monoclonal antibody also in same experiment with comparing.Use α _dPolyclonal antiserum and the dyeing of normal spleen being cut into slices at the monoclonal antibody of CD11c, CD11a, CD11b and CD18 have obtained following result.Use α _dThe observed collection of illustrative plates of polyclonal antiserum does not show the identical collection of illustrative plates that serves as a mark with CD11a, CD11b, CD11c or CD18.Found the marginal zone that some is positioned at white pulp cell the cell of knowing labelling collection of illustrative plates and marginal zone periphery know labelling.This collection of illustrative plates is not observed with other antibody.The individual cells that is scattered in red pulp also is labeled, their possible yes or no and usefulness CD11a and being seen same cell group of CD18 or subgroup.

Really shown some staining cells in the marginal zone with the CD11c labelling, but and α _dPolyclonal antiserum is compared, and antibody does not show the clear ring-type collection of illustrative plates around white pulp, and the labelling in red pulp does not also provide and α _dThe dyeing collection of illustrative plates that polyclonal serum is identical.

Therefore, use α _dThe being seen labelling collection of illustrative plates of polyclonal serum with use at other β ₂It is unique that the being seen collection of illustrative plates of antibody of (CD11a, CD11b, CD11c and CD18) is compared.This explanation, α _dBe different from other β in the intravital distribution of people ₂Integrin.

With monoclonal antibody analyst α _dThe characteristic of expressing

Use hybridoma 169A and the excretory antibody of 169B, with the people α in the immuno-precipitation analysis frozen tissue section _dExpress, with people α in flow cytometry cell line and the peripheral blood leucocyte _dExpression.The hybridoma supernatant that uses in two groups of experiments does not dilute.

Tissue staining

All dyeing is all undertaken by top introduction, except liver slice is undertaken by following method.After acetone fixed, section is at the H that contains 1% ₂O ₂With among the TBS of 1% Hydrazoic acid,sodium salt room temperature cooling 15 minutes.With after the first antibody dyeing, adds a kind of direct and link coupled rabbit anti-mouse antibody of peroxidase, room temperature treatment 30 minutes.Microscope slide washs 3 times in the TBS buffer.Detecting two with a kind of and the anti-rabbit antibody of the direct link coupled pig of peroxidase in 30 minutes at the room temperature incubation resists.Then microscope slide is washed three times in the TBS buffer, add AEC substrate (Vector Labs), develop the color.Microscope slide carries out negative staining with Hematoxylin Gill ' s No.2 (Sigma), and then rinsing in water is dewatered then and counted.

In the spleen section, great majority are expressed and are arranged on the cell that the splenic red pulp identification of morphology is granulocyte and macrophage.A large amount of granulocytes are colored, and macrophage has only a subgroup to produce signal.Sub-fraction trifle dendritic cell in the white pulp are also by α _dAntibody faintly dyes.All detect CD11a and CD18 dyeing at whole red pulp and white pulp.CD11c dyeing is mainly seen in splenic white pulp and the maxicell that is speculated as macrophage in the marginal zone of white pulp, also finds the dispersive dyeing in red pulp simultaneously.CD11b and α _dThe distribution of dyeing in red pulp seems overlapping but inequality, and do not have white pulp to participate in.

Integrin expression in normal and (rheumatoid) arthritis synovial tissue is also compared.In normal structure with all anti-alpha 2 integrin antibodies (comprise can with CD11a, CD11b, CD11c, CD18 and α _dThe antibody of specific immune response) found minimum dyeing, wherein akinete (supposition is a macrophage) extensively distributes.In the synovial membrane of inflammation, the cell of the cluster of the expression multidigit of all integrins around the lymph tubule.α _dWith the expression map of CD11b is similar, as if CD11c expresses not strong, and is limited to a leukocytic subgroup.

In Canis familiaris L., CD11b rather than α _dExpression see Kupffer cell or Kupffer.The dyeing of normal person's liver slice (the same with the introduction of the Canis famililiaris L. section statining of front, above) proved that this dyeing collection of illustrative plates is guarded in the people.In addition, detected low-level CD11c.In the section from a hepatitis patient, the dyeing of all leukocyte integrins all is higher than viewed dyeing in normal hepatocytes, and in these samples α _dBe expressed on macrophage and the granulocyte and be detected.

Use anti-α _dAntibody has been observed minimum dyeing in the section of people's colon, and observes faint smooth muscle dyeing and leukocyte dyeing.High-caliber all leukocyte integrins in section, have been detected from the clone disease patient.

Normal lung has shown the weak α of limited quantity _dPositive cell, they are confirmed as macrophage and neutrophilic granulocyte by form.In the lung tissue of an emphysematic patients, at neutrophilic granulocyte with contain on the macrophage of hemosiderin (a kind of ferrichrome that contains) and detected α _dDyeing, this explanation erythrocyte is by these cytophagies.

The integrin expression that has detected the normal brain activity section and decreased from multiple sclerosis (MS) patient's speckle.In normal brain activity, α _dDyeing is lower than the intensity of CD11a, CD11b and CD11c, and to be confined to morphology and CD68 dyeing typing be the cell of microglia.The CD11b positive cell is positioned at around the tubule, and is distributed in whole tissue.CD11c ⁺As if cell be positioned at tubule, and α _d ⁺Cell is around tubule.In the MS tissue slice, α _dBe expressed on the leukocyte subgroup of microglia and non-macrophage and all be found.α _d ⁺Cell is positioned at speckle to be decreased and whole cortex.α _dThe intensity of signal is identical with CD11c, but is lower than the intensity of CD11b.

With anti-leukocyte integrin and anti-CAM antibody thoracic aorta and ventral aorta section from PDAY (the Atheromatosis Li Shengli determiner of adolescence, LSU medical center) tissue sample are all analyzed.The focus of checking is consistent with aorta fatty striped, gathers with less leukocyte infiltration under the inner membrance of aorta fatty striped by big foam cell (mainly being the macrophage that has the fat of absorption) and forms.Use α _dWith other β ₂Beta 2 integrin alpha chain (CD11a, CD11b and CD11c) adds that single marker research that the special monoclonal antibody of a macrophage labelling (CD68) carries out shows, majority is filled the α that the macrophage of fat is expressed medium level respectively _dAnd CD18, and express low-level or lower CD11a and CD11c than medium level.Only faint expression CD11b, and only by an Expression of Subsets of macrophage.

Carry out double labelling research and determine α _dWith the relative localization of ICAM-R antigen in aorta slice.Because the foam cell in these sections can be dyeed to the special antibody Ham56 of macrophage labelling, and can not therefore can be determined that foam cell is not deutero-by intimal smooth muscle cells by the antibody staining special to smooth muscle actin.Express alpha _dThe male macrophage of CD68 surrounded by the positive leukocyte of little ICAM-R and separately.CD68 is negative but can be by α _dAs if limited with the quantity of the SL of ICAM-R antibody staining.

α _dAs if the distribution in normal structure is at immobilized leukocyte, and the collection of illustrative plates of its distribution collection of illustrative plates and CD11b and CD11c is overlapping but inequality.CD11b and CD11c are that the front has been accredited as the other two kinds of leukocyte beta 2 integrin alpha chains with limited leukocyte distribution.Cellular morphology shows, α _dDyeing is limited to macrophage and granulocyte in a large number, and lymphocyte dyeing is limited.In general, tissue inflammation shows as increases observed quantity of leucocyte and kind in the particular organization, and is attended by the leukocyte integrin and comprises α _dPainted increase.Because the cell of leukocyte integrin is different with spatial distribution in pathological tissue, therefore can know by inference, under different situations, each family member is comprised α _dThere are different functions and part.

What is interesting is α _dExpression in the atherosis in early days focus shows stronger than CD11a, CD11b and CD11c, and this shows α _dIn the foundation of these focuses, may play central role.α _dWith the also column distribution of ICAM-R positive cell, this point is shown α _dAnd interactional evidence is supported between the ICAM-R, and α is described _dMay in these focuses, participate in early stage leukocyte recruitment or activation.

Cell line and peripheral blood leucocyte dyeing

Antibody 169A and 169B can dye to Promyelomonocyte cell line HL60 in FACS.α _dSurface expression in these cells is subjected to the negative effect that PMA stimulates, and reports that PMA stimulates to induce the macrophage differentiation pathway, but do not influenced by DMSO that DMSO induces granulocyte differentiation (Collins etc., " blood " 70 volumes, 1233-1244 (1987)).The FACS collection of illustrative plates that 169A and 169B and PMA stimulate is with opposed with viewed collection of illustrative plates in anti-CD11b and the anti-CD11c monoclonal antibody.A kind of monocytic series THP-1 also shows and can be dyeed a little less than 169A and the 169B.In addition, in FACS, cell subsets of peripheral blood leucocyte medium-sized lymphocyte and mononuclear cell door (gates) shows weak positive, and bone-marrow-derived lymphocyte is found and does not have α _dSurface expression.The lymphocytic CD8 of T ⁺Subgroup is α _d ⁺In addition, antibody 169A and 169B detect antigen in can not cell line below: B cell line JY, Ramos; Basophilic granulocyte is KU813 and T cell line Jurkat, SKW and Molt 16.

According to the result of HL60 cell, granulocyte separates from peripheral blood with the method that the ficoll/hypaque gradient centrifugation is also followed splitting erythrocyte.By in acetic acid, observing nuclear morphology, find that all prepared products＞90% is PMNs.The independent prepared product PMA or 10 of 50ng/ml ^-8M formylated peptide (fMLP) stimulated 30 minutes, made integrin storage release in the possible cell.With respect to a kind of IgG1 contrast, the colony that stimulates does not show low but tangible 169A and the antigenic expression of 169B, observes the raising that can discover after stimulation.In PMNs, α _dMore similar in the HL60 cell with the surface expression ratio of CD11c.Antibody 169B is used to precipitate the heterodimer molecule from a kind of detergent lysate of biotinylated PMNs subsequently, this molecule contain about respectively 150 and 95kD be equivalent to α _dSubunit with the CD18 size.

α _dExistence on PMNs can not be by known relevant dog α _dThe information of expressing is predicted.The dog neutrophilic granulocyte does not resemble its people's homologue, and it expresses t helper cell labelling CD4, also expresses integrin VLA-4, therefore has in Canis familiaris L. and part and function different in the people.

The dyeing of PBL subtribe

This research is used for determining this β ₂The distribution of integrin in human peripheral leucocytes.In addition, also to α _dCell surface density with respect to other β ₂Integrin compares.At last, also to α among the people eosinophilic granulocyte of purification _dThe acute adjusting of expressing is estimated.

Human peripheral leucocytes becomes mononuclearcell level part (containing mononuclear cell, lymphocyte and basophilic granulocyte) and granulocyte (neutrophilic granulocyte and eosinophilic granulocyte) [Warner with density gradient separation, et al., J.Immunol.Meth.105:107-110 (1987)].For some experiment, the eosinophilic granulocyte is purified to purity greater than 95%[Hansel, et al., RImmunol.Meth.122:97-103 (1989) with CD16 immunity magnetic selection].The skin mastocyte separates from application on human skin with the method for enzymolysis, and carries out enrichment [Lawrence, et al., J.Immunol.139:3062-3069 (1987)] by preceding method.

Cell CD11a (MHM24), CD11b (H5A4), CD11c (BU-15) or the α of suitably dilution _d(169A) special monoclonal antibody is carried out labelling.Also use a kind of Mus control antibodies.Washed cell is then with phycoerythrin coupling sheep anti-mouse igg incubation.In some experiments, cell is with the mouse monoclonal antibody or the sheep polyclonal antibody incubation of excessive Mus IgG and FITC-labelling, and above-mentioned antibody is specific to specific cells, and (for example, CD3, CD4 or CD8 are to the T cell-specific; CD16 ⁺Lymphocyte is to the NK cell-specific; Anti--IgE is to basophilic granulocyte special [Bochner, et al., J.Immunol.Meth.125:265-271 (1989)].Use flow cytometry (Coulter EPICS figure) test sample then, use different gatings (gating) to come the identification of cell subgroup.

For the research of using the eosinophilic granulocyte (has detected α in this research _dThe acute rise of expressing), before carrying out labelling by preceding method with different monoclonal antibodies, cell stimulated 15 minutes with Buddhist ripple ester (10ng/ml), RANTES (100ng/ml) [Schall, Cytokine 3:165-183 (1991)] or IL-5 (10ng/ml).

The result shows, α _dOn all peripheral blood eosinophilic granulocytes, basophilic granulocyte, neutrophilic granulocyte, mononuclear cell and NK cell, occur.One group (about 30%) CD8 ⁺Lymphocyte also is found express alpha _dSkin mastocyte and CD4 ⁺Lymphocyte is express alpha not _dIn general, CD11a and CD11b are with than α _dHigh density appears on the lymphocyte, and the latter is with the low relatively horizontal expression that is similar to CD11c.In leukocyte, mononuclear cell and CD8 ⁺Cell has the most highdensity α _d, and the eosinophilic granulocyte has the α of floor level _dExpress.Expression in neutrophilic granulocyte, basophilic granulocyte and NK cell mediates.

Stimulate the periphery eosinophilic granulocyte not cause any β with CC chemotactic factor RANTES ₂The change of integrin expression.Yet handle at CD11b and α with Buddhist ripple ester _dExpression in produced 2 to 3 times increase, but do not influence the expression of CD11a or CD11c.IL-5 handles the selectivity that causes CD11b to express and raises, and does not influence the level of other integrin subunits.

Integrate, these results show, in peripheral blood leucocyte, and α _dGeneral with the horizontal expression suitable with CD11c.The highest expression is found in mononuclear cell and CD8 ⁺A lymphocytic subgroup.The application on human skin mastocyte is express alpha not _dAs if the eosinophilic granulocyte of purification have CD11b and α _dThe Cytoplasm of precursor in depots.But the difference that the relative PMA of IL-5 shows raises explanation, and these depots have nothing in common with each other.

Also use the gating and the surface markers of combination by the introduction of front the dyeing collection of illustrative plates of peripheral blood leucocyte (PBL) subgroup to be measured, come to determine more accurately the lymphocyte populations of 169A/B feminine gender with flow cytometry.PBL separates on Ficoll by preceding method, and use 169A respectively, 169B and at CD14 (monocyte/macrophage labelling), CD20 (B cell), CD56 (NK cell), TXi Baoshouti α/β (T cell), CD16 (neutrophilic granulocyte, NK cell) and α ⁴The antibody staining of (negative marker of neutrophilic granulocyte).Door is determined with size and indicia distribution.

The result shows, at CD14 ⁺Cell in the mononuclear cell door shows low-level 169A and 169B dyeing.Observed bimodal expression map solves with the increase forescatering in the early stage experiment in the lymphocyte door.Blended TCR ⁺/ CD20 ⁺The group shows that have 169A/B lower but consistent level expresses, and one have 50% stained positive, be positioned at the cell mass of slightly high lateral scattering (cell complexity) CD56, shows to have the negative colony of different 169A/B.This feminine gender colony is not TCR, CD20, CD14 or CD16 antibody recognition yet.

α _dSynovial membrane distribute

For determining α _d, other β ₂Integrin and counter receptor thereof are in inflammation and non-inflammation synovial membrane cell distribution not, at different beta ₂The monoclonal antibody of integrin and ig supergene family is used to immunohistology research.Protein expression in normal, osteoarthritis and rheumatoid arthritis synovial tissue samples has obtained mensuration.

The result shows that synovial membrane lining cell layer is expressed high-caliber VCAM-1, CD11b/CD18 and α _d/ CD18.In these cells, CD11c/CD18 expresses and to be restricted, CD11a/CD18 is general detect less than.In the rheumatoid arthritis synovitis, β in synovial cell's layer ₂The increase of the expression of integrin and outgrowth degree are proportional.The cell proportion of expressing CD11c significantly increases, near CD11b and α _dRatio, but the expression of CD11a does not increase.

At the lining lower area of tissue, CD3/CD11a/ICAM-R ⁺Lymphocyticly gather and spread infiltration, intersperse among CD68/CD11b/ α _d ⁺In the macrophage.The remarkable quantity proof of gathering particularly is rich in the strong α in zone at the T cell _dExpress.

The synovial membrane endotheliocyte is differently expressed ICAM-1 and ICAM-2, the evidence that little ICAM-R expresses.

Integrate, these results show that the synovial cell of synovial membrane macrophage and macrophage-like expresses high-caliber β with forming ₂Integrin CD11b and α _dIn synovitis, in lining zone and lining lower area the amplification of this cell subset is arranged all, and be accompanied by the obvious increase that CD11c expresses.High-caliber α also expresses except expressing CD11a and ICAM-R in the lymphocytic specific group of rheumatoid synovial membrane T _d, back a part is presented in the peripheral blood lymphocyte in front with low expression level.

α _dIn ill lung and the expression in the hepatic tissue

To make the 6um slab from the lung tissue of a sarcoidosis individuality with from two individual hepatic tissues of sclerosis, and on Superfrost Plus (VWR Scientific) microscope slide air drying 15 minutes at room temperature.Before use, microscope slide was about 5 minutes of 50 ℃ of incubations.Section is at room temperature fixed 2 minutes in refrigerated (4 ℃) acetone (EM Science), and air drying at room temperature.Section is placed 100ml 1X TBS, 1.1ml 30%H ₂O ₂(Sigma), 1.0 NaN ₃(Sigma) in the solution, placed 15 minutes in room temperature, to remove endogenic peroxidase activity.Each section was at room temperature sealed 30 minutes with the 1X TBS solution that 150 μ l contain 20% normal human serum (Boston Biomedica), 5% normal rat serum (Harlan) and 2%BSA (Sigma).Behind the incubation, blot solution from section lightly.First antibody prepared in lock solution by the protein concentration of 10 μ g/ml, and adds 75 μ l on each tissue slice, room temperature treatment 1 hour.Behind the incubation, section is washed three times in 1X TBS, and each 5 minutes, to remove unconjugated antibody.After the washing, inhale and remove to organize unnecessary TBS on every side the last time.Biotinylated rat anti-mouse antibody (JacksonLaboratories) was diluted to 1: 400 in lock solution, and adds 75 μ l in each section, room temperature treatment 30 minutes.Microscope slide washs 2 times with 1X TBS, each 5 minutes.The link coupled goat-anti biotin antibody of peroxidase (Vector Laboratories) is diluted to 1: 200 in lock solution, and adds 75 μ l, room temperature treatment 30 minutes in each section.Microscope slide washed 5 minutes with 1X TBS washing 2 times at every turn.Add substrate 3-amino-9-ethyl carbazole (AEC) (Vector Laboratories) or diaminobenzidine (DAB) substrate (Vector Laboratories), immerse process color is stopped, microscope slide is negative staining in Gill ' s hematoxylin #2 (Sigma), rinsing in water uses Aquamount (Baxter) or Cytoseal (VWR) to count then.

In the sarcoidosis lung, have only the 217L monoclonal antibody to catch cell, most 217L epi-positions are expressed and are positioned at granuloma.Giant cell in the granuloma is shown as the 217L antigen negative.The expression of 217L epi-position is positioned on the cell that morphology is shown as the epithelial tissue cell, and this cell is the well differentiated phagocyte type cell of macrophage system.In the sarcoidosis lung distribution of other integrins be observed with the distribution of 217L epi-position overlapping, but expression map difference.For example, to cell and giant cell in can the labelling 217L dyeing negative granuloma of the special antibody of every other integrin immunity.。

Express negative from second section that is diagnosed as sarcoid patient for the 217L epi-position.But do not see whether this patient accepted the steroid immunosuppressant from the pathology report, this is the treatment of most common form.

In section from hardened hepatic tissue, anti-α _dFoam cell in the connective tissue between the antibody labeling liver trifle and a lymphocytic subgroup.The distribution of CD11c and α _dExpress overlappingly but different, anti-CD11c antibody is foam cell subgroup of labelling also, but than anti-α _dMore macrophage of antibody labeling and lymphocyte.Distribution and α that CD11a and CD11b express _dExpress significantly not overlapping.

Antibody 217L also can catch phagocyte type cell bunch shape and that separate from the colony of 212D and 217L evaluation.Antibody at CD11b and CD11c is caught 217L in a different manner ⁺Bunch.

In relevant experiment, dye to people's spleen tissue slice and from the series section of inhuman Primate M.nemestrina with antibody 212D and 217L.Also with the flow cytometry evaluation from α in the splenocyte of fresh people and monkey spleen tissue _dExpression.Antibody 212D and 217L can both discern people and monkey splenocyte.With ICC and FACS, α _d ⁺Colony all accounts for the about 20% of total cell, is unlike in the α that shows bigger percentage ratio in the rodent _d ⁺Cell.Positive cell shows and the macrophage homomorphosis.

People's bone marrow stain

People's bone marrow sample is according to the ilium of standard method available from the normal marrow donor.Primary sample in Iscove ' s culture medium by dilution in 1: 3, centrifugal 20 minutes at 2000 RPM.The careful buffy coat of collecting washs once haemolysis in haemolysis buffer (0.83% ammonium chloride, 0.1% sodium carbonate, no EDTA).Cell is resuspended among the PBS that contains 15%FBS, is divided into 100,000 cell/pipes among the 100 μ l, places on ice.Carry out immunostaining by preceding method.In brief, in every hole, add monoclonal mouse-anti α respectively _dAntibody 212D or 217L or mouse-anti people CD18 or mouse-anti people CD50 (ICAM-R is special) antibody is to final concentration 10 μ g/ml, and mixture was incubation on ice 20 minutes.With cell washing twice, and with sheep anti mouse FITC other incubation 20 minutes again.Cell washing twice, and be resuspended in 1% paraformaldehyde.Measure fluorescence with fluorescence-activated cell sorter FACSCAN (Becton Dickinson).

Four result of experiment show α _dSee (intermediate value 27%) on 13 to 43% the cell when expressing, see during with 217L on the cell of 6-55% (intermediate value 21%) with the 212D TPPA, CD18 expresses on the cell that sees 60-96% (intermediate value 71%), and CD50 sees (intermediate value 94%) on the 86-99% cell.

α _dExpression on the breast cancer disease human peripheral blood single nucleus cell

Use the Ficoll partition method from the high risk breast cancer patients'blood sample that has passed through bone marrow transplantation, to separate PERIPHERAL BLOOD MONONUCLEAR CELL.High risk breast cancer patient is the breast carcinoma patient of prognosis mala.Screen express alpha with immunostaining as stated above _dCell.

The result shows, α _dFind on 20% cell when expressing with the 212D TPPA, during with 217L on 13% cell.Antibody 212D has also caught and has shown a subgroup that is likely lymphocytic minicell.Express alpha _dThe percentage ratio of cell suitable with generally observed percentage ratio in the normal blood donor.

In addition, antibody 212D demonstration is not only caught and is CD14 ⁺Maxicell, can also catch experimental identification is CD3 ⁺Much smaller cell.In blood and bone marrow, all observe this result.

Express alpha _dThe variation of the quantity of cell can be used from the variation of the medullary cell composition of different donors extractions and explain (for example bone marrow quantity is compared with circulation blood quantity).

Embodiment 19

α _dThe rise of expressing

Because the leukocyte integrin generally raises [Rabb during observed immunity changes in hemodialysis and in the chronic renal failure, et al., J.Am.Soc.Nephrol.6:1445-1450 (1995) and Rabb, et al., Am.J.Kidnet Dis.23:155-166 (1994)], we have detected α in hemodialysis and chronic renal failure _dThe surface expression of/CD18.In addition, also studied α after PKC stimulates _d/ CD18 is in external expression.

Whole blood sample is available from 5 inpatients that do not have nephropathy that select at random.Before padding and flow cytometry, the PMA of blood sample and 50ng/ml was 37 ℃ of incubations 30 minutes.Simultaneously also from the patient who suffers from chronic renal failure, collect blood sample.Patient is stable, does not have diabetes, time carries out hemodialysis on every Wendesdays.Baseline sample is obtained before beginning dialysis, then 15 minutes and 180 minutes draw sampless in a cuprophane film dialysis.Use from the blood sample of the normal individual of not suffering from known disease in contrast.

For carrying out cell dyeing, with the antibody 169A of 5 μ g and 169B (with a negative control 1B7) and 100 μ l whole bloods incubation 15 minutes in the dark.In each mixture, add Becton Dickinson lytic reagent (2ml), in the dark continued incubation 10 minutes.Sedimentation cell is resuspended in PBS then.Centrifugal sedimentation cell once more, and mix with FITC link coupled two is anti-, in the dark incubation is 30 minutes.Then with cell with PBS washing, centrifugal, abandoning supernatant, be resuspended in 1.0% formalin.

Use Rabb, et al.[J.Am.Soc.Nephrol.6:1445-450 (1995)] method carry out flow cytometry.Sample is analyzed on FACscan flow cytometry (Becton Dickinson) with Simulset software (Becton Dickinson).22,000 cells of the minimum analysis of each sample.Granulocyte, mononuclear cell and lymphocyte subgroup carry out the branch door with forward light scattering and lateral light scattering.The purity of cell subsets is estimated with CD45 dyeing and CD14 dyeing.

The result shows, α _dThe expression of/CD18 can detect from the blood that normal person's donor extracts, be expressed on the mononuclear cell the highest, minimum on lymphocyte.In the expression on the neutrophilic granulocyte between mononuclear cell and lymphocyte.Be weaker than dyeing with antibody 169B dyeing with antibody 169A.PMA handles and raises α _d/ CD18 expresses, particularly on neutrophilic granulocyte and mononuclear cell.

In sample from person having renal failure, before beginning dialysis, α _dBeing expressed on neutrophilic granulocyte, mononuclear cell and the lymphocyte of/CD18 detects.Using the dialysis of leukocyte activation film after 15 minutes, detect α _dA small amount of increase that/CD18 expresses.In the later stage of handling, in fact the expression on mononuclear cell and the lymphocyte reduces.This result shows, α _d/ CD18 expresses with observed dialysis back CD11a/CD18, CD11b/CD18 and L-and selects plain expression different.

Embodiment 20

The separation of rat cdna

Because dog and people α _dSubunit all exists, and we manage to comprise rat (present embodiment), mice at other species, and (embodiment 20, above) the middle homologous genes that separates.

Demonstration and people α from a rat spleen λ gt10 library (Clontech), have been obtained _dThe partial sequence of the rat cdna of dna homolog.This library is pressed 2 * 10 ⁴The pfu/ plate is laid on 150mm LBM/ agar plate.By standard method [Sambrook, et aL, Molecular Cloning:alaboratory manual, introduction p.2.110] is offered (Amersham) on the Hybond_ film with the library, degeneration 3 minutes, in and 3 minutes, with buffer washing 5 minutes.Film is placed on the Stratalinker (Stratagene) immediately, make DNA crosslinked with automatically cross-linked device.Film respectively under low and height stringent condition, is carried out prehybridization and hybridization in 30% or 50% formaldehyde.Earlier with a kind of ³²The probe of P labelling screens film, and this probe is from people α _dPrepare among the cDNA, and be equivalent to clone the base 500 to 2100 of 19A2 (SEQ ID NO:1).The program that probe uses Boehringer Mannheim ' s random primer test kit to recommend according to manufacturer is carried out labelling.Filter membrane washs at 55 ℃ with 2 * SSC.

Identified two clones, they are named as 684.3 and 705.1, and they show and people α _d, people CD11b and people CD11c sequence homology.Two clones are at 3 of gene ' zone and people α _dGene coupling concerning 684.3, originates in base 1871 and extends to base 3012, to cloning 705.1, is base 1551 to 3367.

For separation comprises 5 ' regional more complete rat sequence, use same program in the first screening, (see embodiment 20, above), identical library is screened once more but use by a kind of Mus probe of cloning the A1160 preparation.Select single, isolating plaque from programmed screening, on the LBM/ agar plate, be maintained single clone.In the PCR of standard program, use sequencing primer 434FL and 434FR (being respectively SEQ ID NO:34 and 35) to prepare the DNA that is used to check order.

5′-TATAGACTGCTGGGTAGTCCCCAC-3′ (SEQ?ID?NO：34)

5′-TGAAGATTGGGGGTAAATAACAGA-3′ (SEQ?ID?NO：35)

The DNA that PCR produces carries out purification with the Quick program that post (Qiagen) recommends according to manufacturer that spins.

Identified two clones, they are named as 741.4 and 741.11, and they are 684.3 and 705.1 overlapping with the clone, in the overlapping region, clone 741.4 and 741.1 with clone 684.3 and 705.1 100% homologies.A merging with people α _dGene has the rat cdna of homology and lists in SEQ ID NO:36, and the aminoacid sequence of prediction is listed in SEQ ID NO:37.

Rat α _dThe clone of 5 ' end

Use a Clontech rat spleen RACE clone test kit, the program according to manufacturer is recommended has obtained a rat α _dGene 5 ' cDNA fragment.The oligonucleotide of the gene specific that uses is named as 741.11#2R and 741.11#1R (being respectively SEQ ID NO:59 and 58).

5′-CCAAAGCTGGCTGCATCCTCTC-3′ (SEQ?ID?NO：59)

5′-GGCCTTGCAGCTGGACAATG-3′ (SEQ?IN?NO：58)

Oligonucleotide 741.11#2R comprises the 131-152 base pair among the SEQ ID NO:36, but direction is opposite; 741.11#1R comprise the 696-715 base pair among the SEQ IN NO:36, direction is also opposite.Use 3 '-most oligonucleotide 741.11#1R carry out first PCR.Then the DNA that produces with oligonucleotide 741.11#2R and first set reaction carries out the PCR second time.On 1% agarose gel, detected a band of about 300 base pairs.

The product program of recommending according to manufacturer of PCR is connected among the plasmid pCRTAII (Invitrogen) with the second time.Picking white (positive) clone, and join 100 μ l and contain among the LBM of M13 K07 phage culture of the 50mg/ml Carbenicillin stock solution of 1 μ l and 1 μ l, and join respectively in the single hole of a round bottom 96 hole tissue culturing plates.Mixture was 37 ℃ of incubations 30 minutes to 1 hour.After the first incubation period, add the LBM (containing the 50mg/ml Carbenicillin stock solution of 1 μ l and 1: 250 dilution of 10mg/ml kanamycin stock solution) of 100 μ 1, continue to be incubated overnight at 37 ℃.

Use 96 aseptic mesoporous metals to shift suction nozzle, the supernatant of 96 orifice plates is transferred on 4 Amersham Hybond_ nylon leaching films.According to standardization program with filter membrane degeneration, neutralization and crosslinked.Filter membrane at 20ml prehybridization buffer (5 * SSPE, 5 * Denhardts, 1%SDS, 50 μ g/ml denatured salmon sperm dnas) 50 ℃ of prehybridization a few hours and shake.

Oligonucleotide probe 741.11#1 and 741.11#1R (being respectively SEQ ID NO:56 and 57) comprise base pair 86-105 (SEQ ID NO:36), are respectively forward and reverse, they

Carry out labelling by following method.

5′-CCTGTCATGGGTCTAACCTG-3′ (SEQ?ID?NO：56)

5′-AGGTTAGACCCATGACAGG-3′ (SEQ?ID?NO：57)

With 12 μ l dH ₂About 65WP oligonucleotide DNA among the O be heated to 65 ℃ two minutes.The 10mCi/ml γ of adding 3 μ l in test tube- ³²P-ATP, and add 4 μ l 5X kinase buffer liquid (Gibco) and 1 μ l T4 DNA kinases (Gibco).Mixture was 37 ℃ of incubations 30 minutes.After the incubation, add the oligonucleotide probe of every kind of labelling of 16 μ l in the prehybridization buffer and on the filter membrane, and continue hybridization at 42 ℃ and spend the night.Filter paper at room temperature washs in 5 * SSPE, 0.1%SDS three times, and each 5 minutes, and radioactive automatic developing 6 hours.With the positive colony amplification, and the program purify DNA that uses Magic Mini Prep test kit (Promega) to recommend according to manufacturer.Clone 2F7 is selected to check order, and shows with clone 741.11 that in the overlapping region 100% homology is arranged.Complete rat α _dNucleotide sequence is listed in SEQ IDNO:54, and its aminoacid sequence is listed in SEQ ID NO:55.

The feature of rat cdna and aminoacid sequence

Rat β ₂The nucleic acid of beta 2 integrin alpha subunit and aminoacid sequence are former all not to be had to report.But with the people β that has reported ₂The sequence of beta 2 integrin alpha subunit comparison shows that, the rat clone who is separated to and the aminoacid sequence and the α of its prediction _dNucleotide and aminoacid sequence are the most closely related.

In nucleic acid level, isolating rat cdna clone and people α _dThe homogeneity of demonstration 80% when cDNA compares shows 68% homogeneity when comparing with people CD11b, the homogeneity of demonstration 70% when comparing with people CD11c, the homogeneity of demonstration 65% when comparing with Mus CD11b.When comparing, there is not tangible homogeneity with people CD11a and Mus CD11a.

At amino acid levels, by the prediction rat polypeptide that separates the cDNA coding with people α _dWhen comparing, polypeptide shows 70% homogeneity, when comparing, show 28% homogeneity with people CD11a, when comparing, show 58% homogeneity with people CD11b, when comparing, show 61% homogeneity with people CD11c, when comparing, show 28% homogeneity, when comparing, show 55% homogeneity with Mus CD11b with Mus CD11a.

Embodiment 21

The α of rat tissue _dThe Northern that expresses analyzes

Extracting RNA from one group of Lewis rat tissue comes with a kind of rat α _dProbe carries out Northern and analyzes.Sample comprises the total RNA from normal spleen, kidney, liver, lung and bone marrow, adds from normal spleen, brain, spinal cord, thymus, skin, small intestinal, rat antigen activated T cells, the spleen of suffering from EAE disease (experimental allergic encephalomyelitis) and the poly (A of lymph node ⁺) RNA.The technology that experiment uses embodiment 6 to introduce is carried out.

α _dProbe is selected from a zone of rat cdna, comprises the 1184-3008 nucleotide among the SEQ ID NO:54, and this Regional Representative and rat CD11c and CD11b have the zone of the homology of minimum level.This 1124bp probe is by digesting 10 μ g rat α with the EcoRI restriction endonuclease _dCDNA clone 684.3 prepares.This fragment gel-purified, and be used for the reaction of random primer labelling by the introduction among the embodiment 6.The Northern trace is undertaken by prehybridization, hybridization and the washing introduced among the embodiment 6, except probe presses 5.5 * 10 ⁵Cpm/ml joins in the hybridization buffer.

Behind the radioactive automatic developing 5 days, containing the total RNA of spleen and from normal mice and from the poly (A of the spleen of suffering from active EAE rat ⁺) detect band in the RNA swimming lane, in the rat that suffers from active EAE, the amount of RNA is obviously greater than the RNA amount from normal mice.The size of detected transcript is big or small consistent with total length rat cdna clone's.

Embodiment 22

Rodent α _dSpecific antibody-at rat α _dThe preparation and the feature analysis of the antibody of I domain/Hu IgG4 fusion rotein

Because reference β ₂The I domain of integrin participates in the part combination, can infer rat α _dAlbumen also is same.Therefore to rat α _dThe special monoclonal antibody of I domain immunity has α _dIn the rat model in conjunction with the human disease who participates in is useful.

Oligonucleotide " rat α-DI5 " (SEQ ID NO:87) and " rat α-DI3 " (SEQ INNO:88) are from rat α _dPrepare in the sequence, they are equivalent to base pair 469-493 and base pair 1101-1125 (rightabout) among the SEQ ID NO:54 respectively.The PCR that oligonucleotide is used for a standard reacts, and prepares one and contains the rat α that leap is equivalent to the I domain of base pair 459-1125 among the SEQ ID NO:54 _dDna fragmentation.The program that the PCR product is recommended according to manufacturer is connected among the carrier pCRTAII (Invitrogen), selects a positive colony and amplification, and (Chatswoth, GA) Midi Prep test kit is according to the program purify DNA of manufacturer's recommendation with a Qiagen.DNA, with the band of one 600 base pair of gel-purified and is connected in the pDCS1/HuIgG4 expression vector subsequently with XhoI and BglII enzymic digestion in the reaction of a standard.Select a positive colony amplification, with a Qiagen MaxiPrep test kit purify DNA.

COS is laid on the 100mm culture dish by partly converging, at 37 ℃ at 7%CO ₂Middle grow overnight.The DMEM rinsing of cell usefulness 5ml once.In 5ml DMEM, add the rat α that 50 μ l DEAE-glucosans, 2 μ l chloroquines and 15 μ g introduce previously _dI domain/HuIgG4DNA.Mixture is added the COS cell, 37 ℃ of incubations 3 hours.Shift out culture medium then, in accurate 1 minute, add the 10%DMSO among the 5ml CMF-PBS.Cell leniently washs once with DMEM.In cell, add the DMEM that 10ml contains 10%FBS, continue at 37 ℃ at 7%CO ₂In be incubated overnight.Second day, replace culture medium with fresh culture medium, and continue to cultivate again other 3 days.Collect culture medium, and in plate, add fresh culture medium.After three days, regather culture medium, discard plate.Repeat said process, up to collecting 2 liters of culture supernatant.

A ProsepA_ post (Bioprocessing) and by the following purifying protein of introducing on the supernatant that will collect by preceding method.

Earlier with the lavation buffer solution column scrubber that contains 35mM Tris, 150mM NaCl, pH7.5 of 15 times of volumes, with supernatant be lower than 60 column volumes/hour the low rate upper prop.Behind the upper prop, wash post with 50mM citric acid, the pH5.0 of 0.55M diethanolamine, pH8.5 and 15 times of volumes of the lavation buffer solution of 15 times of volumes, 15 times of volumes.Albumen 50mM citric acid, pH3.0 eluting.Albumen neutralizes with 1.0M Tris, pH8.0, and dialyses in aseptic PBS.

Rat α _dThe I domain protein is analyzed with the method that embodiment 14 introduces.Detected albumen is to move with the observed same way as of people I domain protein.

Preparation is at rat α _dThe monoclonal antibody of I domain/HuIgG4 fusion rotein

Mice use respectively in advance isopyknic Freund's complete adjuvant (FCA) (Sigma) in the rat α of emulsive 50 μ g purification _dI domain/HuIgG4 fusion rotein carries out immunity.About 200 μ l antigen/adjuvant prepared products are expelled to 4 sites of every mouse back and sidepiece.After two weeks, mice is by injecting 100 μ l emulsive rat α in isopyknic incomplete Freund (FIA) in advance _dI domain/HuIgG4 antigen (50 μ g/ Mus) carries out booster immunization.After other two weeks, mice carries out booster immunization with 50 μ g antigens among the 200 μ l PBS through vein.

For estimating the serum titer in the immune mouse, at immune back 10 days for the third time animal is carried out getting blood behind the eye socket, make haemagglutination behind the eye socket, and centrifugalize serum.The immunoprecipitation that serum is used for the rat spleen cells of biotin (BIP) labelling.Serum from every mice can both immunoprecipitation rat α _dProtein band with the expection molecular weight of rat CD18.Select a mice to merge, and undertaken the 4th time by the method for introducing in the booster immunization for the third time and strengthen.

The hybridoma supernatant screens with the antibody capture method by following introduction.(Dynatech, Cambridge Massachusetts) are used in sheep anti mouse IgA, the IgG or the IgM (Organon Teknika) that dilute at 1: 5000 among 50mM sodium carbonate buffer, the pH9.6 at 4 ℃ by 50 μ l/ holes and wrap quilt the Immulon_4 plate.Plate washs three times with the PBS (PBST) that contains 0.05%Tween_20, and adds 50 μ l culture supernatant.After 30 minutes, press the preceding method washing at 37 ℃ of incubations.Be added among the PBST horseradish peroxidase of dilution in 1: 3500 sheep anti-mouse igg 9 (Fc) (Jackson ImmunoResearch, West Grove, Pennsylvania).Plate carries out incubation by preceding method, and washs four times with PBST.And then, adding contains 1mg/ml o-phenylenediamine (Sigma) and 0.1 μ l/ml 30%H ₂O ₂100mM citric acid, the substrate of pH4.5.15% sulphuric acid that adds 50 μ l after 5 minutes stops color reaction.Read to read on the plate instrument absorption at 490nm place at Dynatech.

Also use immobilized rat α from the supernatant in the hole of containing antibody _dI domain/HuIgG4 fusion rotein carries out elisa assay.An ELISA who carries out with the plate of HuIgG4 antibody sandwich is as the contrast at the reaction of IgG fusion partner.Select positive hole on the rat spleen cells lysate, further to screen with BIP with the technology of introducing below.

Rat α _dThe preparation of I domain/HuIgG4 fusion rotein polyclonal antiserum

At rat α with 100 μ g purification in the Freund's complete adjuvant (FCA) _dBefore I domain/HuIgG4 fusion rotein carries out immunity, two rabbits are taken a blood sample in advance.Per three weeks are used the same dose duplicate injection in the incomplete Freund (IFA).After three injections, to the rabbit blood sampling that experimentizes, and the standard immunoassay that the serum of collecting is used on the rat spleen cells lysate precipitates.The result determines, from the serum of two rabbits all can with rat α _dImmunoreation.100 μ g antigens among the reuse IFA carry out booster immunization to rabbit, and the serum and the rat α that collect with immunoprecipitation analysis _dImmunoreactive raising.Give animal last booster immunization after 10 days, serum is collected in blood-letting.

Rat α _dThe histology

Pressing the technology of introducing among the embodiment 18 will be at rat α _dThe rabbit polyclonal serum of " I " domain preparation is used for the dyeing of rabbit tissue slice.Dyeing collection of illustrative plates in the section of freezing and paraffin-embedded rat spleen with use α at the people _dThe viewed collection of illustrative plates of antibody is identical, and painted individual cells is distributed in whole red pulp.This dyeing collection of illustrative plates with use at the observed collection of illustrative plates of the monoclonal antibody of rat CD11a, CD11b and CD18 different.In addition, in thymus, observed the positive staining collection of illustrative plates that individual cells is distributed in whole cortex.When these are organized in the dyeing of the rabbit anteserum before the immunity, can not produce any signal.

Antibody specificity is analyzed

Rat is used CO ₂The execution that suffocates is taken out spleen with the surgical technic of standard.Spleen was touched a metallic screen with a 3cc syringe plug collected splenocyte in 20mlRPMI.In cell harvesting to one 50 a tapered test tube, and in suitable buffer, wash.

Cell washs three times in cold D-PBS, and with 10 ⁸To 10 ⁹Cell density be resuspended in 40ml PBS.The NHS-Biotin (Pierce) that adds 4mg in cell suspension, reaction at room temperature continues accurately to carry out 15 minutes.Sedimentation cell, washing is three times in cold D-PBS.

Cell is by 10 ⁸Cell/ml is resuspended in (1%NP40,50mMTris-HCl, pH8.0,150mM NaCl, 2mM CaCl in the cold lysis buffer ₂, 2mM MgCl, 1: 100 pepstatin, leupeptin and press down the enzyme peptide solution, before adding cell, add; 0.0001 gram PMSF crystal adds before adding cell).With lysate vibration rotation 30 seconds, room temperature incubation 5 minutes, and incubation on ice 15 minutes.10,000g made the insoluble matter precipitation in centrifugal 10 minutes with lysate.Supernatant is collected a new pipe, be stored between 4 ℃ to-20 ℃.

1ml lysate and 200 μ l protein A Sepharose_ slurries (Zymed) are incubated overnight at 4 ℃ carry out pre cleaning.The lysate of pre cleaning divided to install to by 50 μ l/ pipe be used for every kind in the Eppendorf pipe and detect antibody.25 μ l polyclonal serums or 100 to 500 μ l monoclonal antibody supernatant are added in the pretreated lysate, and the mixture that obtains was rotated incubations 2 hours at 4 ℃.The bonded 100 μ l rabbit anti-mouse iggs (Jackson) of protein A Sepharose_ pearl among adding and the PBS continue to rotate incubation 30 minutes in room temperature then.The slight centrifugal pearl precipitation that makes, and wash buffer (10mM HEPES, 0.2M NaCl, 1%Trition X-100_) with cold wash and wash three times.The sucking-off supernatant.Add 20 μ l and contain 2 * SDS sample buffer of 10% beta-mercaptoethanol.Sample boiled in water bath 2 minutes.With 5%SDS PAGE gel on the sample, after the separation, albumen spent the night with constant current to be transferred on the nitrocellulose membrane.Nitrocellulose room temperature sealing 1 hour, discards the sealing buffer with the TBS-T that contains 3%BSA.Be added in the streptavidin-HRP conjugate (Jackson) of dilution in 1: 6000 among the 0.1%BSA TBS-T, continued incubation 30 minutes in room temperature.Filter membrane is with TBS-T washing three times, and each 15 minutes, and carry out radioactive automatic developing according to the program of manufacturer's recommendation with Amersham ' s ECL test kit.

At total length rat α _dProteic MONOCLONAL ANTIBODIES SPECIFIC FOR

Purification of rat α from rat spleen cells _dPrepare and be used to produce the Mus α of the Chinese People's Anti-Japanese Military and Political College _dThe immunogen of monoclonal antibody.Collect from the spleen of the normal female Lewis rat in about 50 12-20 age in week, and by making its metallic screen that passes through a precision from single cell suspension of tissue preparation.Containing 150mM NH ₄CL, 10mM KHCO ₃, 0.1mM EDTA, the cracking of pH7.4 buffer remove erythrocyte, remaining leukocyte is with phosphate-buffered salt (PBS) washed twice.The centrifugation splenocyte, and containing 50mM Tris, 150mM NaCL, 2mM CaCl ₂, 2mM MgCl ₂, 10mM PMSF, leupeptin, pepstatin and 1%Triton X-100_ buffer in cracking.The splenocyte cracking is by every ml lysis buffer 5 * 10 ⁸Splenocyte carried out on ice 30 minutes.The centrifugal insoluble matter of removing.

CD11a, CD11b and CD11c remove from the spleen lysate with immunoprecipitation by following method.Protein A-Sepharose_ the slurry and the anti-rat immune globulin of 2mg rabbit that with volume are 750 μ l were 4 ℃ of incubations 30 minutes.Anti-Mus-the protein A of rabbit-Sepharose_ washs three times with lysis buffer, and is suspended in the lysis buffer that final volume is 1.5ml.Every kind of rat β with about 200 μ g ₂Special monoclonal antibody 515F (rat CD11a is special), OX-42 (rat CD11b is special) and the 100g (rat CD11c is special) of integrin adds in the rat spleen lysate of each 50ml., after 30 minutes the anti-Mus-protein A of the rabbit of 500 μ l-Sepharose_ is joined in the spleen lysate at 4 ℃ of incubations, put upside down and be rotated in 4 ℃ of mixing 30 minutes.Lysate was precipitated and the anti-Mus-protein A of the rabbit-bonded CD11a of Sepharose_, CD11b and CD11c at 2500 * g in centrifugal 10 minutes, supernatant is gone to a clean 50ml centrifuge tube.Repeat to precipitate twice in addition to guarantee to remove fully CD11a, CD11b and CD11c with antibody 515F, OX-42 and 100g.

Remaining β in the lysate ₂Integrin separates with affinity purification method.About 250 μ l and the Mus CD18 of the link coupled Chinese People's Anti-Japanese Military and Political College of CHBr-Sepharose_ monoclonal antibody 20C5B slurry are joined in the lysate, put upside down rotation at 4 ℃ and mixed 30 minutes.Made antibody/antigen complex precipitation in centrifugal 10 minutes at 2500 * g, precipitation is stored in 4 ℃ then with lysis buffer washing three times.The immunity of armenian hamster

1.6-8 the armenian hamster in age in week is earlier with 50 μ g emulsive rat α that contains in Freund's complete adjuvant _dThe recombiant protein that I domain and human IgG 4 heavy chains merge carries out immunity.Then be used in emulsive rat α in the incomplete Freund after the initial immunity at 14,33 and 95 days _dI domain/HuIgG4 carries out immunity.And then carry out twice independent fusion, they are named as 197 and 199.

Merging 197 preceding 4 days (306 days), give the rat α of purification from splenocyte to a hamster _dAlbumen and rat α _dThe merging thing of the Chinese hamster ovary celI of transfection.Merging the rat α of first three day (307 days) with purification _dAlbumen and α _dThe Chinese hamster ovary celI of transfection carries out booster immunization.Rat α _dThe Chinese hamster ovary celI of transfection is by following method preparation.

Rat α with a coding total length _dProteic gene is inserted in the pDC1 carrier, and electrifies with a people CD18-pRC construct one and to be transformed in the Chinese hamster ovary celI.Transfectional cell exists under the hypoxanthic condition growth with the cell of pRC construct of having selected successfully transfection, and exists under the condition of G418 growth with the cell of pDC1 construct of having selected transfection.After three weeks, cell rat α _dSpecial rabbit polyclonal serum dyes, and classifies with FACS.Collect and express high-caliber surperficial α _dThe cell (account for greatly sum 3%) of a little percentage ratio, and further amplification.Repeat FACS and select to provide for several times α _dThe cell mass of high level surface expression.

α _dCells transfected is also used a kind of rat α with flow cytometry _dSpecial polyclonal serum and the special monoclonal antibody TS1.18.1 of a kind of people CD18 analyze.The result proves, the high-caliber rat α of the expressing cho cell of transfection _dWith people CD18.

At last, α in the cell _dEstimate with immunoprecipitation with the expression of CD18.A kind of rat α _dSpecial rabbit polyclonal serum is found the albumen of two kinds of different molecular weights of immunoprecipitation: high-molecular-weight protein is about 170kD, and low molecular weight protein (LMWP) is about 95kD.These are found and transfection CHO cell surface rat α _dThe expression unanimity of/people CD18 heterodimer complex.

Merging that day, take out spleen, to be organized in the frosted end that is immersed in two microscope slides among the serum-free RPMI1640 that adds 2mM L-glutamic acid, 1mM Sodium Pyruvate, 100 units/ml penicillin and 100 μ g/ml streptomycins grinds, the suspension (RPMI) of formation individual cells (Gibco, Canada).Cell suspension filters 70 aseptic sieve Nitex cell filters (Becton, Dickinson, Parsippany, New Jersey).200 * g washing in centrifugal 5 minutes 2 times.The precipitation that is obtained is suspended in 20ml serum-free RPMI again.The thymocyte cell that takes out from three Balb/c children Mus also uses the same method and is prepared.Before fusion, will (RPMI Utah) maintains the NS-1 myeloma cell of exponential phase for HycloneLaboratories, Logan with containing 10%Fetalclone serum (FCS) merging first three day, at 200 * g centrifugal 5 minutes, precipitation was with the method washed twice of introducing previously.

With about 1.15 * 10 ⁸Splenocyte and 5.8 * 10 ⁷The NS-1 mixing with cells, centrifugal and sucking-off supernatant.Knock test tube and take out cell precipitation, and in 1 minute, add the PEG1500 (among 75mM Hepes, the pH8.0 50%) (Boehringer Mannheim) of 37 ℃ of 7ml, and stir.In 7 minutes of following, add other 14ml serum-free RPMI.The RPMI that adds 8ml again.Cell centrifugal 10 minutes at 200 * g.Abandoning supernatant.The precipitation be suspended in again 200ml contain 15%FBS, 100mM hypoxanthine sodium, 0.4mM aminopterin, 16mM thymus pyrimidine (HAT) (Gibco), 25 units/ml IL-6 (Boehringer Mannheim) and 1.5 * 10 ⁶The RPMI of thymocyte cell.Suspension divides in 10 the 96 flat tissue culturing plates in hole (Corning, the United Kingdom) by 200 μ l/ holes.The the 4th, 5, the 6 and 7 day cell of feeding after fusion, method are with the about 100 μ l of a 18G pin (Becton Dickinson) sucking-off from every hole, by the method for introducing previously except not containing the thymocyte cell bed board culture medium that adds 100 μ l/ holes.

After fusion the 10th day, from the supernatant that merges the hole with Flow cytometry itself and rat α _dThe reactivity of the Chinese hamster ovary celI of/people CD18 transfection.With about 5 * 10 ⁵Rat α _dThe Chinese hamster ovary celI of transfection is resuspended among the RPMI that 50 μ l contain 2.0%FBS and 0.05% Hydrazoic acid,sodium salt, adds in the about 100 μ l hybridoma supernatant in the 96 hole circle base plates.Painted positive control comprises the anti-α of rabbit _dPolyclonal serum and TS1/18 (anti-people CD18).Cell was incubation on ice 30 minutes, at FACS buffer (RPMI, 2.0%FBS, 0.05% Hydrazoic acid,sodium salt) in washing three times, and with a kind of in the FACS buffer final dilution be that 1: 200 the link coupled goat-anti hamster of FITC antibody (Jackson ImmunolResearch Labs) was incubation on ice 30 minutes.Cell washs three times in the FACS buffer, is resuspended in the FACS buffer of 200ml.Sample is analyzed with Becton Dickinson FACscan analyser.For guaranteeing that positive colony is rat α _dSpecial, with the Chinese hamster ovary celI repetition screening process of non-transfection.To meet and rat α _dCHO transfectional cell reaction and not with the hole clone of the Chinese hamster ovary celI reaction normal of non-transfection.

After the first screening, from the cell in positive hole with double dilution method and then in the RPMI that contains 15%FBS, 100mM hypoxanthine sodium, 16mM thymus pyrimidine and 10 units/ml IL-6 limiting dilution clone.In the limiting dilution step, determine to show the percentage ratio in the hole of growing, clone's property is predicted with the Poisson distribution analysis.The hole that shows growth after 10-12 days with facs analysis.After the last clone, increase in RPMI and 11%FBS in positive hole.The clone has produced these standards of usefulness and has been judged as male hole, four the independent sub-clones that increased from this hole, called after 197A-1,197A-2,197A-3 and 197A-4 respectively.

Carry out 199 merge before, second hamster at 307 days with 2.3 * 10 ⁶Rat α _d(RAD) Chinese hamster ovary celI of transfection carries out booster immunization.In fusion preceding 4 days (334 days) with merge preceding 3 days (335 days) and carry out last twice immunity.334 days booster immunization comprises 2 * 10 ⁶Rat α _dThe bonded purification of rat α of the Chinese hamster ovary celI of transfection and 200 μ l and Sepharose_ (front introduction) _dThrough the intraperitoneal administration.335 days booster immunization comprises 5 * 10 ⁶Rat α _dThe Chinese hamster ovary celI of transfection is also through the peritoneal injection administration.199 fusions of merging are merged identical with screening process and 197.Identify and cloned three supernatant and rat α _dThe hybridoma of reaction, called after 199A, 199H and 199M.

2. use the identical process of 197 and 199 fusions to carry out the immunity second time.Behind 334 days booster immunizations, before 394 and 395 days, do not carry out further immunity.Before fusion, give 2 * 10 through intraperitoneal to hamster ⁶The Chinese hamster ovary celI of RAD transfection and 300 μ l purification of rat α _d-Sepharose_.The fusion of the fusion of then carrying out 205 is identical with fusion 199 and 197 with screening process, and except in the clone, armenian hamster ELISA reagent such as goat-anti armenian hamster antibody (Jackson ImmunolResearch Labs) are used as first screening.Then screen with FACS with the positive hole that the method is identified by introducing.Merge 205 and produce three independent positive colonies, called after 205A, 205C, 205E.

3. prepare the Mus α of the Chinese People's Anti-Japanese Military and Political College at another kind _dIn the monoclonal antibody method, the BALB/c mouse in 6 to 12 ages in week was at the 1st day rat α with purification _d-Sepharose_ carries out immunity through subcutaneous administration in Freund's complete adjuvant.Carried out second time booster immunization with the identical immunogen in the incomplete Freund through identical approach at the 25th day.Carry out at the 42nd day with for the second time identical booster immunization for the third time.No longer further strengthen before the booster immunization before fusion, booster immunization comprises the rat α of 400 μ l (merging 226) and 250 μ l (merging 236) purification before merging _dThe peritoneal injection of-Sepharose_.Every volume comprises about 10 to 15 μ g antigens, and this is determined by coomassie dyeing.For merging 226, strengthen carrying out before merging at 62 days and 63 days, merge and carried out at 66 days; For merging 236, strengthen carrying out before merging at 132 days and 133 days, merge and carried out at 136 days.The difference that two kinds of fusion processs and the armenian hamster of introducing previously merge used process is that the splenocyte that uses 5: 1 ratios by comparison, in armenian hamster merges, uses 2: 1 ratio to the NS-1 cell.Other aspects of fusion process are identical with the armenian hamster process.

Fusion 226 is identical with the process that merges 197,199 and 205 with clone's process with 236 screening, except carrying out the elementary screening with ELISA.In ELISA, use the full molecule of a kind of sheep anti mouse to come from the hybridoma supernatant, to catch murine antibody.And use a kind of sheep anti mouse horseradish peroxidase thing to detect murine antibody.Positive colony then screens according to the introduction of merging 197 to 205 with FACS.

Merge 226 and produce 9 positive colonies, called after 226A, 226B, 226C, 226D, 226E, 226F, 226G, 226H and 226I.Merge 236 and produce 10 positive colonies, called after 236A, 236B, 236C, 236F, 236G, 236H, 236I, 236K, 236L and 236M.The monoclonal anti body and function ELISA that produces from these clones carries out typing according to the introduction of embodiment 15, finds that all antibody is the IgG1 isotype.

At rat α _dThe feature analysis of monoclonal antibody

For analyzing the Mus α of the Chinese People's Anti-Japanese Military and Political College _dThe feature of monoclonal antibody, biotin labeled spleen lysate is prepared by the introduction among the front embodiment 12 part D.Lysate carries out pre cleaning before being used for immunoprecipitation.At first, the normal mice immunoglobulin of 50 μ g/ml is joined in the lysate, the solution that is obtained is put upside down rotation at 4 ℃ and was mixed 30 minutes.Add the protein A-Sepharose_ that is coated with the anti-rat immune globulin of rabbit and starch 75 μ l, continue to put upside down rotation and mixed 30 minutes.In a benchtop microcentrifuge 4 ℃ with 15, centrifugal 5 minutes of 000rpm makes the protein A pearl precipitation of the anti-Mus of rabbit bag quilt, collects supernatant.Discard deposit.

Hybridoma to each clone, the supernatant of about 300 μ l is added an Eppendorf microcentrifugal tube, add pepstatin, leupeptin and but the biotinylation rat spleen lysate of enzyme peptide 100X stock solution, 100 μ g PMSF crystal and 50 μ l pre cleanings of Triton X-100_, the 30 μ l of 30 μ l 30% therein.With the sample rotation of leniently vibrating, place one to put upside down rotation rotary head 30 minutes at 4 ℃.A control sample is used in and adds the 10mg/ml rabbit Mus α of the Chinese People's Anti-Japanese Military and Political College in the 50 μ l rat spleen lysates _dSpecific polyclonal antibody prepares.

Through behind 30 minutes incubations, in each sample, add the PBS slurry of 75 μ l protein A-Sepharose_ pearls, put upside down the rotation incubation 30 minutes at 4 ℃.Protein A-link coupled pearl was precipitated at 4 ℃ with 15,000 in a benchtop microcentrifuge in centrifugal 5 minutes, collected supernatant.Sedimentary pearl is washed in proper order with the detergent of following a series of 1ml: the buffer #1 that contains 10mM Tris, 400mM NaCl, 1.0%Triton X-100_, pH8.0; The buffer #2 that contains 10mM Tris, 400mM NaCl, 1.0%Triton X-100_, 0.5%Triton X-100_, pH8.0; The buffer #3 that contains 10mM Tris, 400mM NaCl, 0.1% deoxycholic acid, pH8.0; Contain 10mM Tris, 400mM NaCl, 0.5%M LiCl ₂, pH8.0 buffer #4.Last washing is carried out with buffer #1.In each washing,, and precipitate with benchtop microcentrifuge with pearl rotational oscillation leniently.Supernatant is removed with the transfer aspirator.After the last washing, remaining buffer is shifted out from pearl with the Hamilton syringe.In each precipitation, add 50 μ l and contain the SDS sample buffer that bromophenol blue and pyronin Y dyestuff and final concentration are 10% beta-mercaptoethanol.With mixture thermal agitation rotation 1-2 minute, and room temperature incubation 5-10 minute.Sample 4 ℃ in benchtop microcentrifuge with 15, centrifugal 5 minutes of 000rpm, the albumen that collect to discharge is transferred in the new pipe.Five equilibrium thing from each sample boiled in water bath 4 minutes, went up the 7.5%SDS-PAGE gel then.After PAGE separates, albumen was transferred on the nitrocellulose membrane in the 200mAmps transfer in 1 hour.And filter membrane spent the night 4 ℃ of sealings in 3.0%BSA/TBS-T solution.Add the 0.1%BSA-TBS-T solution of the streptavidin-OPD that contains dilution in 1: 6000 to every filter paper, continued incubation 1 hour in room temperature.Filter membrane washs in TBS-T 5 times, and each 10 minutes, the program of recommending according to manufacturer with Amersham ' s ECL test kit developed the color.

Clone 199M is found can a kind of heterodimer albumen of immunoprecipitation.Bigger protein protomer has the molecular weight of about 170-175kD, with the usefulness rabbit Mus α of the Chinese People's Anti-Japanese Military and Political College _dThe albumen size of polyclone contrast immunoprecipitation is consistent.Also precipitated second kind of albumen that molecular weight is about 95kD, consistent with the molecular weight of CD18.

Embodiment 23

The specificity of monoclonal antibody 199M

Use coupling to have the CNBr-Sepharose_ affinity column of 199M monoclonal antibody to come affinity purification rat α from the splenocyte lysate _dIn brief, with about 1.3 * 10 ¹⁰Rat spleen cells cracking in the buffer that contains 150mM NaCl, 10mM PMSF, 10mM Tris, 1%Triton X-100_, pH8.0.Cell in the buffer incubation 30 minutes on ice and 4 ℃ with about 10,000 * g centrifugal 30 minutes.

With antibody 199M as follows with the activatory Sepharose_4B of CNBr-(Pharmacia) coupling.The activatory resin of 1 gram was suspended among the 1mM HCl 15 minutes,, contains 0.1mM HCO with 15ml with 15ml 1mM HCl washing three times ₃, 0.5M NaCl, pH8.0 coupling buffer washing once.Antibody 199M in the coupling buffer is joined in the resin suspension by the about 10-20mg/ml of final concentration, and mixture is incubated overnight at 4 ℃.Second day, centrifugal link coupled resin precipitated, the abandoning supernatant of making.Untreated group by sealing at room temperature incubation in 0.1M Tris, pH8.0 in 1 hour on the resin.Link coupled resin washs with 0.1M citric acid, pH3.0, and is stored in the lysis buffer that contains 0.1% Hydrazoic acid,sodium salt with the form of 1: 2 slurry.

For carrying out affinity purification, splenocyte and the link coupled agarose resin of the 199M of 0.4ml put upside down mixing spends the night carries out incubation.The centrifugation resin is used 15ml lysis buffer washing resin 4 times then.The gel of every part of about 100 μ l is boiled rapidly in the reduced form sample buffer that contains 0.1M Tris-HCl, pH6.8,2.0%SDS, 20% glycerol, 0.0002% bromophenol blue, 10% beta-mercaptoethanol (final concentration is 5%), and go up sample, albumen is differentiated with 6.0% polyacrylamide sds gel (SDS-PAGE).

When SDS-PAGE separated, the material of affinity purification was found and contains two main and less important albumen kinds.Major protein band with 90kD molecular weight is consistent with the known dimensions of CD18, this band is not checked order.Second main band to 160kD detects this band and α _dThe predicted molecular weight unanimity.In addition, also having detected apparent molecular weight is the less important band of 200kD.160kD and 200kD use albumen aminoterminal order-checking to analyze, and with the result with use rat α _dThe aminoacid sequence of cDNA prediction and the aminoacid sequence of known CD11c and CD11b compare.The sequence of 160kD and 200kD band is found all and uses cloned rat α _dThe consensus amino acid sequence of prediction, this shows the α that has two kinds of forms _d, they may be splice variants or be caused by glycosylation difference.

Embodiment 24

Use the rat express alpha _dThe T cell proliferation test of macrophage

Separation is from the express alpha of rat spleen _dMacrophage be used as the myelin basic protein specificity T cell line/lineage that antigen presenting cell (APC) stimulates a kind of LR-21 by name.In brief, rat (BioMag, Cambridge is MA) through intravenous injection with the ferrum granule of 100 μ l volumes.Second day, prepare single cell suspension with spleen, have the particulate α of the ferrum of engulfing with the magnetic collection _d ⁺Macrophage.Flow cytometry and immunoprecipitation show, the cell 50 of engulfing ferrum is to 80% being α _d ⁺

The result shows, express alpha _dSplenic macrophage than other APC for example the thymus macrophage be very weak APC.Also in proliferation test, use α _dMale macrophage and LR-21 cell line have detected the monoclonal antibody of called after 205C.Carry out proliferation test then as follows.

α _dExpress male splenic macrophage by 6 * 10 ⁶The density of cell/ml is suspended among the RPMI that contains 5% normal rat serum, adds the macrophage suspension of 100 μ l in every hole.Cell from LR-21 cell line presses 1 * 10 ⁶The density of cell/ml is suspended among the RPMI that contains 5% normal rat serum, and adds 50 μ l suspensions in every hole.The monoclonal antibody 205C that in every hole, adds 50 μ l volumes to final concentration be 50,10 and 2 μ g/ml.Plate is 37 ℃ of incubations 72 hours, and adds 1uCi ³The H-thymus pyrimidine carries out last 24 hours incubations.Cell harvesting to fiberglass packing, is measured with direct β enumerator (Packard Matrix96) ³H integrates.

Experimental result shows that the antibody 205C of high concentration (10 and 50 μ g/ml) can reduce the T cell proliferation in dosage dependence mode.

Embodiment 25

α from rat marrow _dImmunoprecipitation

Method with PBS flushing femur is collected medullary cell from a Lewis rat.Cell is pressed method washing, biotinylation and the immunoprecipitation that embodiment 18 introduces basically, uses the monoclonal antibody of 20 μ g purification to come immunoprecipitation albumen from the cell lysate of 100 μ l pre cleanings.The proteic detection of immunoprecipitation is undertaken by the method for introducing previously.

Rat α _dMonoclonal antibody 205C immunoprecipitation is with two bands of 160kD and 95kD migration, so Da Xiao band and carrying out the observed α of proteinic immunoprecipitation with same antibody from the splenocyte lysate _dThe α of/CD18 and β chain big or small consistent.Antibody mediated immunity precipitation and the α of the Mus CD11a of the Chinese People's Anti-Japanese Military and Political College, CD11b or CD11c _dDifferent α chains, all antibody be a kind of molecular weight of co-precipitation albumen consistent with the known molecular amount of CD18 all.

Embodiment 26

α _dExpression in animal model

Preliminary result shows, rat α _dOptionally be expressed in the subgroup of macrophage, comprise the subgroup and the immobilized bone marrow macrophage of cortex macrophage in the thymus, the Kupffer Cell in the liver, the dimension solencyte among the central nervous system, peritoneal macrophages.In addition, sulfur sugar salt macrophage subgroup is stimulating the back to show α with dexamethasone _dUp-regulated.Viewed rat α _dThe restricted expression of macrophage show and need do further to analyze the expression in the several animal models.

Rat α _dExpression in the phenylhydrazine model

Giving phenylhydrazine to animal can cause a large amount of erythrocyte (rbc) damage and produce temporary anemia.Impaired erythrocyte is removed from circulation by the red pulp macrophage, thereby causes tangible splenomegaly.Express alpha by inference _dMacrophage may participate in impaired erythrocyte and other allogenic materials and from circulation, remove.

For verifying this hypothesis, organize rat more and handle with saline or the phenylhydrazine that is dissolved in the saline separately, and carry out administration by the 100mg/kg body weight through intraperitoneal.In some experiments, rat is used at rat α _d" domain I " polyclonal antiserum of producing handle.

Different time points after the phenylhydrazine administration is put to death animal, the parameter that spleen is heavy and hematocrit is removed as erythrocyte.In addition, collection kidney, spleen regulating liver-QI carry out histopathological analysis, comprise CD11a, CD11b, CD11c and α _dCarry out immunostaining.

Total discovery shows, with phenylhydrazine (saline control and α _dHandle) to handle after 4 days, rat has produced rapid splenomegaly, and hematocrit reduces.Use α _dPolyclonal serum is handled inductive splenocyte weight of phenylhydrazine or not influence of blood specific volume decline.

The tissue of the rat that will handle from saline and 4 days phenylhydrazines is made the 4um slab, and on Superfrost Plus (VWR Scientific) microscope slide dry 15 minutes of air at room temperature.Before the use, with microscope slide about 5 minutes of 50 ℃ of incubations.Section is fixed 2 minutes in room temperature in cold (4 ℃) acetone (EM Science), and in drying at room temperature.Section is placed the 1X TBS of 100ml, the H of 1.1ml 30% ₂O ₂(Sigma), 1ml 10%NaN ₃(Sigma) place 15 minutes to remove endogenic peroxidase activity in room temperature.Each section contains the solution of 30% normal rat serum (Harlan Bioproducts) among the 1X TBS, 2%BSA (Sigma) room temperature sealing 30 minutes with 150 μ l, then leniently from the removal solution of cutting into slices.Each section is diluted to the biotinylated hamster Mus α of the Chinese People's Anti-Japanese Military and Political College of 13.3 μ g/ml in confining liquid with 75 μ l _dAntibody 205C was room temperature treatment 1 hour.Behind the incubation, section is washed three times in 1X TBS, and each 5 minutes, to remove any unconjugated antibody.Is organizing on every side unnecessary TBS sucking-off the washing back the last time.The link coupled goat-anti biotin antibody of peroxidase (Vector Laboratories) is diluted to 1: 200 in confining liquid, 75 μ l are used for each section, following 30 minutes of room temperature.Behind the incubation, microscope slide washs 2 times in 1X TBS, each 5 minutes.Add AEC (Vector Laboratories) substrate, colour developing is stopped by immersing.With microscope slide negative staining in Gill ' s hematoxylin #2 (Sigma), and rinsing in water, dehydration successively in 70%, 95%, 100% ethanol and dimethylbenzene then.Section cytoseal (VWR) sealing then.

In rat spleen section with saline treatment, most α _dExpression is arranged on the cell that splenic red pulp morphology is accredited as macrophage, granulocyte and a lymphocytic subgroup.And in the rat spleen that phenylhydrazine is handled, splenic red pulp has experienced morphological change, makes that the cellular type of unique evaluation is to have engulfed impaired erythrocytic big macrophage group.The great majority of the macrophage that these are huge are observed express alpha _dIn the rat that phenylhydrazine is handled, also show express alpha in the splenic white pulp _dThe quantity of macrophage increase.

On the Rats Spleen that phenylhydrazine is handled, also carried out double-label experiment and determined α _dExpression with CD11c.As previously mentioned, α _dBe expressed in the splenic red pulp and show to have engulfed in the impaired erythrocytic big macrophage and detect.CD11c expresses and also detects in the big macrophage in splenic red pulp, and α is compared in the demonstration of CD11c positive cell in red pulp _dPositive cell is many.Most of express alpha _dMacrophage also express CD11c, although observe sub-fraction α _dPositive macrophage is not expressed CD11c.The positive macrophage of CD11c that a colony also arranged is express alpha not _d

Therefore immune analysis shows, CD11c's is expressed in the 4th day with saline control and compares and show and raise in the spleen of the animal that phenylhydrazine is handled.And other integrins CD11a, CD11b and α _dExpression show the influence not handled by phenylhydrazine.With polyclone " I " domain α _dAntibody treatment also shows does not influence the red pulp macrophage to erythrocytic picked-up, is α but engulf erythrocytic macrophage majority _dPositive.Engulf impaired erythrocytic α _dPositive macrophage is not present in the spleen that the phenylhydrazine administration collects after 7 days.

Use the apoptosis test subsequently and the Rats Spleen of phenylhydrazine processing in 4 days is carried out double-label experiment with the link coupled antibody 205C of biotin.The tissue of the rat that will handle from normal rat and 4 days phenylhydrazines is made 4 microns slabs, goes up dry 15 minutes of air at room temperature at Superfrost Plus (VWR Scientific), and is stored in-20 ℃.Before the use, microscope slide is heated to 50 ℃.The microscope slide of heating is placed the 1X TBS that contains 100ml, the H of 1.1ml 30% ₂O ₂(Sigma), 1ml 10%NaN ₃(Sigma) buffer is placed 15 minutes to remove endogenic peroxidase activity in room temperature.Each section contains the solution of 20% normal rat serum (Harlan Bioproducts) among the 1X TBS, 2%BSA (Sigma) 37 ℃ of sealings 10 minutes with 150 μ l, then leniently from the removal solution of cutting into slices.Each section is diluted to the biotinylated hamster Mus α of the Chinese People's Anti-Japanese Military and Political College of 26.6 μ g/ml in confining liquid with 75 μ l _dAntibody 205C was 37 ℃ of incubations 30 minutes.Cut into slices then and in 1X TBS, wash three times, each 5 minutes, to remove unconjugated antibody.Is organizing on every side unnecessary TBS sucking-off the washing back the last time.The link coupled avidin/biotin complex of alkali phosphatase (Vector Laboratories) that will prepare according to the indication of manufacturer is used for each section, and 37 ℃ following 20 minutes.Behind the incubation, microscope slide washs 2 times in 1X TBS, each 5 minutes.Section is fixed 5 minutes at 4 ℃ with paraformaldehyde (Sigma).Cut into slices then and in 1X PBS, wash, and place CSK buffer (100mM NaCl, 300mM sucrose, 10mM PIPES pH6.8,3mM MgCl ₂, 0.5%Triton-X-100_) in 4 ℃ two minutes.To cut into slices in 1X PBS room temperature rinsing 2 minutes, section was with 1X PBS washing three times, each 5 minutes then.(Boehringer Mannheim) is applied to each section with the TUNEL reactant mixture, and 37 ℃ following 60 minutes.Behind the incubation, section is washed three times in 1X PBS, each 5 minutes.The principle of apoptosis test kit is similar in situ hybridization, TUNEL reagent (FITC is link coupled) and " otch " DNA hybridization.To change son-POD (the link coupled antibody of peroxidase of the FITC sign on a kind of TUNEL of identification reagent) and be used for each section, 37 ℃ following 30 minutes.Section is washed three times with 1X PBS, each 5 minutes.Add AEC (Vector Laboratories) substrate, immerse color development stopping in the water.Section Aquamount (VWR) sealing.

In model, a large amount of cells in the red and white marrow zone of spleen all produce apoptosis, but have engulfed erythrocytic big macrophage (express alpha in the red pulp _dAnd disappeared at the 7th day of model) find to produce apoptosis.

Rat α on the normal rat spleen _dThe cell type analysis of expressing

For determining which kind of rat cell type express alpha _d, on the normal rat spleen, carry out double labelling dyeing.The section for preparing the normal rat spleen by preceding method is sealed step until rat blood serum.After adding the first cell marking antibody, the link coupled sheep anti-mouse antibody of alkali phosphatase (JacksonLaboratories) is diluted to 1: 500 at the identical diluent that is used for first antibody, and 75 μ l is joined in each section room temperature 30 minutes.Microscope slide washs 2 times in 1X TBS, washs 5 minutes at every turn.The anti-goat-anti body of the donkey of alkali phosphatase enzyme mark (JacksonLaboratories) is diluted to 1: 300 in the antibody dilution agent, and 75 μ l are added to each section, room temperature 30 minutes.After washing as stated above and sealing, it is the biotinylated hamster Mus α of the Chinese People's Anti-Japanese Military and Political College of 20 μ g/ml that 75 μ l protein concentrations are accepted in each section _dAntibody (205C), each 1 hour 45 minutes, unconjugated antibody was removed in washing then.Last washing back is inhaled on every side from tissue and is removed unnecessary TBS.The link coupled goat-anti biotin of peroxidase (VectorLaboratories) is diluted to 1: 200 in the antibody dilution agent.And 75 μ l are added each section, following 45 minutes of room temperature.Microscope slide washed 5 minutes with 1X TBS washing 2 times at every turn.Add AEC substrate (Vector Laboratories), immerse color development stopping in the water.Add Fast Blue substrate (Vector Laboratories) then, immerse color development stopping in the water.Microscope slide Aquamount (Baxter) sealing then.

The double antibody immunocytochemistry is used at CD5, CD2, CD4, CD8, NK labelling or HIS45 (a kind of T cell marking) in conjunction with a kind of anti-α _dAntibody carries out, to manage to determine express alpha _dThe phenotype of cell.Use CD2/ α _dOr HIS45/ α _dDo not detect double labeling cells.In the splenic red pulp of normal rat, α _dCan labelling be found the minicell bunch that also can express CD5.Do not determine also whether this CD5 positive cell is T cell or B cell.A small set of α in red pulp _dThe determined CD4 that also expresses of express cell.In addition, a subgroup of NK cell and CD8 positive cell also is accredited as express alpha _dTherefore, the α in the spleen _dExpression is found in a subgroup of a subgroup, NK cell of T cell, possible B cell and a subgroup of macrophage.α _dFrom the expression on huge granulocytic leukocyte (LGL) tumor cell of F344 rat model

Use in the F344 rat and set up a LGL leukemia rat model available from the tumor cell of National Cancer Institute, this tumor cell is by in intravenous injection to the 3 male F344 rat (every 1,000,000 cell).Disease shows needs three months, at this moment, with several sacrifice of animal, checks tissue with FACS and tissue chemical analysis.

For carrying out facs analysis, take out the part spleen, prepare single cell suspension by the introduction of the following examples.In brief, the spleen tissue is cut into pieces with pincers, and exists under the condition of D-PBS by a metallic screen.Centrifugation cell, and be resuspended in 30ml D-PBS.In a 15ml centrifuge tube, 5.0ml cell suspension is laid on 5.0ml Histopaque and prepares Histopaque gradient (Sigma).With this gradient with Beckman desk centrifuge centrifugal 30 minutes at 1500rpm, and collecting cell layer, washing is once counted with hematimeter in D-PBS.Cell is resuspended in FACS buffer (RPMI-1640/2%FBS, 0.2% Hydrazoic acid,sodium salt), and making its density is 1 * 10 ⁶Cell/sample.

Cell by with the link coupled hamster of the biotin Mus α of the Chinese People's Anti-Japanese Military and Political College _dThe antibody incubation of Chinese People's Anti-Japanese Military and Political College's Mus cell marking of antibody 205C (10 μ g/ml) and a series of FITC labellings carries out two tone dyeing.These second kind of antibody comprises anti-macrophage-FITC, anti-CD3-FITC and anti-IgM (B cell)-FITC antibody (all available from PharMingen), adds the antibody (Harlan) of the link coupled NK of the having cell-specific of a kind of FITC.The link coupled antibody of FITC all uses by 10 μ l/ samples.

Sample earlier on ice with 205C-biotin antibody incubation 30 minutes, washing is three times in the FACS buffer, is resuspended in the FACS buffer of 1.0ml.The FITC conjugate is added with 5 μ l streptavidin-PE (Pharmigen), and sample was placed 30 minutes on ice.Behind the incubation, sample washs three times in the FACS buffer, is resuspended in 200 μ l FACS buffer.With Becton Dickinson FACscan test sample, and with Lysis II software (BectonDickinson) analytical data.

The result has proved α overwhelmingly _dExpression at NK or LGL cell surface.The cell of B cell and T cell marking stained positive does not show α _dExpress, show the α that slight extent is only arranged with the painted cell of macrophage labelling _dExpress.It is believed that on this point of disease, spleen mainly is made up of the NK tumor cell, this and observed jumpbogroup splenocyte are by NK labelling and α _dExpress painted unanimity as a result.

These observe also consistent with the result who uses peripheral blood cells of collecting from same animal and the parallel laboratory test of carrying out FACS by top method.Use the result of peripheral blood cells to show that circulation NK cell is express alpha also _d, and the cell of other cell markings of expression does not show α in the blood _dExpress.But use the result of peripheral blood cells and be owing to change violent the splenocyte result who exists the percentage difference of different cell types to produce in spleen and the peripheral blood unlike supposition.

When using rat spleen cells from these animal patterns then to carry out FACS, obtained identical result.When the cellular preparations of face was further analyzed in the use, the LGL tumor cell also showed the dyeing that CD18 and CD11a, CD11b and CD11c express.

The ICC program that use is introduced has above been carried out tissue chemical analysis to normal and NK F344 diseased tissue.Preliminary data show, α _dReach at NK F344 tumor lung and hepatic tissue invading the exterior, but in corresponding normal structure, do not detect or with utmost point low expression level.The disease lung tissue shows α _dAt little and big cell cluster and be scattered on the individual cells of whole lung and express.NK F344 liver shows around the tubule and scatters weak labelling on other cells of whole tissue.At other β ₂The antibody of integrin shows that these molecules are expressed with similar level, though labelling collection of illustrative plates difference in normal structure and diseased tissue.

In parallel analysis, normal rat thymus shows slight α in the cell dispersion of cortex _dExpress, and F344 NK thymus shows α _dExpression increases.Normal spleen shows the expression in the red pulp, and the NK spleen has bunch shape labelling of the whole tissue that distributes.

Begin to detect the NK spleen weekly from seizure of disease then, the result shows α _dExpression improve, up to the 3rd week, around the, descend then.

Embodiment 27

Use anti-α _dMonoclonal antibody suppresses the cracked analysis of target cell of NK tumor cell induction

A specific function of NK cell is targeting and kills and wounds virus infected cell and foreign cell.For analyzing the ability of NK lysis particular target cell.Target cell is used ⁵¹The chromium labelling when cracking takes place, can detect the radioactivity of rising in culture medium.The previous verified α that on the NK cell, expresses _d, it may participate in the cell killing of NK cell-targeting by inference.For estimating this hypothesis, tumor cell and α _dα is analyzed in antibody precincubation _dEffect in a functional analysis.

α _dThe preparation of positive NK-tumor effect cell

With the NK tumor cell injection F344 rat that obtains from National Cancer Institute, and before taking out spleen, in animal, went down to posterity for 3 to 4 weeks.Take out spleen, cut into pieces, in the presence of D-PBS, pass through a metallic screen.With the cell suspension that obtained on the Beckman desk centrifuge room temperature centrifugal 10 minutes with 1500rpm.The sucking-off supernatant is resuspended in 30mlD-PBS with cell.

Come the Histopaque of the mononuclearcell of autoblood to separate then as follows.In 6 15ml centrifuge tubes, add 5ml Histopaque (Sigma), spread the cell suspension 5ml that one deck is introduced above thereon.Cell on the Beckman desk centrifuge room temperature centrifugal 30 minutes with 1500rpm.The collecting cell layer merges, and counts with hematimeter.The dilution of several isolating tumor cells of preparation in D-PBS, and be the Mus α of the Chinese People's Anti-Japanese Military and Political College of 50 μ g/ml existing or not having concentration subsequently _dIncubation under the condition of antibody.Control antibodies comprises a kind of Chinese People's Anti-Japanese Military and Political College Mus CD18 antibody and a kind of Mus ICAM-1 of Chinese People's Anti-Japanese Military and Political College antibody, and these two kinds of antibody are also with concentration and the cell incubation of 50 μ g/ml.Before test, tumor cell and antibody were about 30 minutes of 37 ℃ of precincubation.

The chromium labelling of Yak-1 target cell

Yak-1 cell (ATCC) is an a kind of mouse lymphoma cell system, with this kind cell culture in 10%FBS/RPMI 1640.Centrifugal collecting cell presses about 1 * 10 in 1.5 to 4.0ml RPMI " test media " ⁷Cell density resuspended.Test media prepares with 500ml RPMI1640,5ml Pen-Strep antibiotic solution and 10ml FBS.In Yak-1 cell suspension, add about 200 to 300uCi's ⁵¹Chromium slightly mixed incubations 45 to 60 minutes at 37 ℃ then.Behind the incubation, volume is increased to 50ml with test media, and centrifugation cell.Abandoning supernatant in 1 to 3ml test media, and is adjusted to 5 * 10 with density with cell suspension ⁴Also preparing concentration is 1 * 10 ⁷The non-marked Yak-1 cell of cell/ml is as own control.

By measure the activity of labeled cell with the labeled cell suspension of 100 μ l in three repetitions of gamma counter test.

The short-term chromium-release test

Each dilution of NK effector lymphocyte by in three 100 μ l volume test media that repeat to be laid in the 96 hole micro plates, is added the Yak-1 cell of 100 μ l labellings in every hole.The release that self is spontaneous or background obtains at each effector lymphocyte's dilution factor incubation with 100 μ l labeled cells and non-marked Yak cell.Always ⁵¹Chromium discharges and to add 1.0%Triton in the target cell be used in a series of holes and obtain.Spontaneous release is with finding in the hole that target cell is only arranged ⁵¹The chromium amount is measured.The incubation of effector lymphocyte/target cell carried out 4 hours at 37 ℃, and is then that plate is centrifugal, collects 100 μ l supernatant from every hole, measures radioactivity.

Lysis is active calculates with following formula:

The result shows, the Mus α of the hamster Chinese People's Anti-Japanese Military and Political College _dAntibody (comprising 199M and 205C) and mouse anti rat α _dAntibody (226A, 226B, 226C, 226D, 226F, 226G, 226H and 226I) all can not influence the ability of the target cell of NK tumor cytotoxicity or cracking labelling.

Embodiment 28

Mice cDNA clone's separation

Manage to separate mice α _dCongener

Crisscrossing between the probe that uses two kinds of PCR to prepare is planted: be equivalent to the 1.5kb fragment that the people clones the base 522 to 2047 of 19A2 (SEQ ID NO:1); With one be equivalent to the 1.0kb rat fragment that the people clones base 1900 to 2900 bases of 19A2 (SEQ ID NO:1).The use of people's probe is listed in SEQ ID NO:38 and 39 respectively and is called the primer of ATM-2 and 9-10.1 to being prepared with PCR; The rat probe uses the primer of listing in SEQ ID NO:34 and 35 respectively that 434L and 434R are prepared.Sample carries out 30 circulation temperature step orders: 94 ℃ then 94 ℃ of incubations 4 minutes; 50 ℃ 2 minutes; 72 ℃ 4 minutes.

5′-GTCCAAGCTGTCATGGGCCAG-3′ (SEQ?ID?NO：38)

5′-GTCCAGCAGACTGAAGAGCACGG-3′ (SEQ?ID?NO：39)

The program 200uCi[that the program purification that the PCR product is recommended according to manufacturer with Qiagen Quick Spin test kit, about 180DNA use Boehringer Mannheim random primer labelling test kit to recommend according to manufacturer ³²P]-dCTP carries out labelling.Uncorporated isotope uses Centri-sep spin post, and (NJ) program of recommending according to manufacturer is removed for Princeton Separation, Adelphia.Before the use, probe is with 0.2N NaOH degeneration, with 0.4MTris-HCl, pH8.0 neutralization.

Bed board is carried out by the every 15cm flat board of about 30,000 plaques in mouse thymus oligomerization dT-primer cDNA library (Stratagene) among the λ ZAP_II.Nitrocellulose membrane (Schleicher ﹠amp; Schuell, Keene, NH) plaque on is offered thing and stirred incubation 1 hour at 50 ℃ in the prehybridization solution that contains 30% Methanamide (8ml/ offers thing).The people and the rat probe of labelling are added in the prehybridization solution, continue to be incubated overnight at 50 ℃.Filter membrane in room temperature washed twice in 2 * SSC/0.1%SDS, is washed once in 2 * SSC/0.1%SDS at 37 ℃, in 2 * SSC/0.1%SDS, wash once at 42 ℃.Filter membrane on KodakX-Omat AR film at-80 ℃ with screen exposure 27 hours.

Will 4 plaques of positive signals line on the LB culture medium flat plate that contains magnesium (LBM)/Carbenicillin (100mg/ml) again in the thing offering of duplicating, and be incubated overnight at 37 ℃.Plaque is offered with Hybond_ filter membrane (AmershaM), by first method for screening probe in detecting, and on Kodak X-Omat AR film at-80 ℃ with screen exposure 24 hours.

12 plaques that provide positive signal are gone to contain 10mM Tris-HCl and 1mMMgCl ₂Low Mg ⁺⁺In the phage diluent.Use T3 and T7 primer (being respectively SEQ IN NO:13 and 14) and following reaction condition to measure the size of inserting son with PCR.Sample carries out 30 circulation temperature step orders then 94 ℃ of incubations 4 minutes: 94 ℃ 15 seconds; 50 ℃ 30 seconds; 72 ℃ 1 minute.

It is the different band of 300 bases to 1kb that 6 samples produce magnitude range.By discharging phasmid with the helper phage coinfection, and recirculation prepares Bluescript_SK ^-(Stratagene).What obtained is cloned in overnight incubation in the LBM/ Carbenicillin (100mg/ml).(Madison, WI) separate by the program of recommending according to manufacturer with Promega Wizard_miniprep test kit for DNA.The EcoRI restriction enzyme analysis of purify DNA has proved the molecular weight that detects with PCR.Inserting DNA checks order with the primer M13 and the reverse .1 of M13 that list in SEQ ID NO:40 and 41 respectively.

5′-TGTAAAACGACGGCCAGT-3′ (SEQ?ID?NO：40)

5′-GGAAACAGCTATGACCATG-3′ (SEQ?ID?NO：41)

Order-checking is undertaken by the introduction among the embodiment 4.

In 6 clones, two clones that only are called as 10.3-1 and 10.5-2 provide sequence information, and are identical 600bp fragments.This 600bp sequence and people α _dRespective regions 68% homogeneity is arranged, with people CD11a 40% homogeneity is arranged, with people CD11c 58% homogeneity is arranged, with mice CD11b 54% homogeneity is arranged.Then this 600bp fragment is used for separating the possible mice α of coding _dThe more complete cDNA of congener.

Mice spleen cDNA library among the λ ZAP_II (Stratagene) (oligomerization dT and random primer) pressed about 2.5 * 10 ⁴Plaque/15cmLBM flat board carries out bed board.Plaque is offered on the Hybond_ nylon transfer membrane (Amersham),, neutralize with 0.5M Tris alkali/1.5M NaCl/11.6 HCl, and wash with 2 * SSC with 0.5M NaOH/1.5M NaCl degeneration.Method with ultraviolet radiation is linked to DNA on the filter membrane.

The probe 10.3-1 and the 10.5-2 of labelling screen about 500,000 plaques in advance by method mentioned above in use.Probe added in the prehybridization solution and at 50 ℃ be incubated overnight.Filter membrane washs once in 2 * SSC/0.1%SDS at 37 ℃ in room temperature washed twice in 2 * SSC/0.1%SDS, washs once in 2 * SSC/0.1%SDS at 42 ℃.Filter membrane on Kodak X-Omat AR film at-80 ℃ with screen exposure 24 hours.14 are used for second and take turns screening duplicating the plaque of offering to provide on the thing positive signal, second takes turns screening screens basic identical with the first round, except increasing the washing of last high stringent condition, the washing of high stringent condition is, at 50 ℃ in 2 * SSC/0.1%SDS, 50 ℃ in 0.5 * SSC/0.1%SDS, at 55 ℃ in 0.2 * SSC/0.1%SDS.Filter membrane on Kodak X-OmatAR film at-80 ℃ with screen exposure 13 hours.

18 positive plaques are transferred to low Mg ⁺⁺In the phage diluent, and measure the size of inserting son with pcr amplification by top introduction.7 samples provide the These positive bands that scope is 600bp to 4kb.The EcoRI restriction enzyme analysis of purify DNA has proved the observed size with PCR, and DNA is reverse with primer M13 and M13.1 (being respectively SEQ ID NO:40 and 41) checked order.

A clone who is named as B3800 contains one and is equivalent to people α _dThe 4kb in the zone of 5 ' end downstream 200bp of 19A2 clone inserts son, and comprises 553 bases of 3 ' untranslated region.Clone B3800 shows and people α _dRespective regions 77% homogeneity is arranged, with the respective regions of people CD11a 44% homogeneity is arranged, with the respective regions of people CD11c 59% homogeneity is arranged, with the respective regions of mice CD11b 51% homogeneity is arranged.Second clone A1160 is insertion of a 1.2kb, and this inserts son and people α _dAbout 12 nucleic acid places, 5 of coding region ' end initial methionine downstream are arranged side by side.Clone A1160 shows and people α _dRespective regions 75% homogeneity is arranged, with the respective regions of people CD11a 46% homogeneity is arranged, with the respective regions of people CD11c 62% homogeneity is arranged, with the respective regions of mice CD11b 66% homogeneity is arranged.

Fragment is more cloned the clone A1160 of 5 of 19A2 ' end near the people, length is 1160 bases, has one section overlapping region that originates in base 205 and be extended to base 1134 with clone B3800.Clone A1160 has the insertion (the base 704-814 of clone A1160) of non-existent 110 bases in the overlapping region of B3800.This insertion may be positioned at exon-intron join domain [Fleming, et al., J.ImmunoL 150:480-490 (1993)], removes before being connected subsequently of clone A1160 and B3800.

The mice α that infers _dThe rapid amplifying of 5 ' cDNA end of clone

Use RACE PCR[Frohman, " RACE:Rapid Amplification of cDNAEnds; " in PCR Protocols:A Guide to Methods and Applications, Innis, et al. (eds.) pp.28-38, Academic Press:New York (1990)] obtain to lose infer mice α _d5 ' terminal sequence of clone comprises 5 ' non-translated sequence and initial methionine.The program of recommending according to manufacturer use a mice spleen RACE-Ready test kit (Clontech, Palo Alto, CA).Designing primer two antisenses, gene specific---A1160 RACE1-is elementary to carry out elementary and nested PCR with A1160 RACE2-nested type (SEQ ID NO:42 and 43).

5′-GGACATGTTCACTGCCTCTAGG-3′ (SEQ?ID?NO：42)

5′-GGCGGACAGTCAGACGACTGTCCTG-3′ (SEQ?ID?NO：43)

Primer SEQ ID NO:42 and 43 corresponds respectively to from the initial zone of 5 ' end, 302 and 247 bases.The mice spleen cDNA that uses 5 ' anchor primer (SEQ ID NO:44) and test kit to provide carries out PCR by method mentioned above.

5′-CTGGTTCGGCCCACCTCTGAAGGTTCCAGAATCGATAG-3′

(SEQ?ID?NO：44)

PCR product electrophoresis showed the band of about 280 bases of size, it is cloned the program sub-clone that test kit (Invitrogen) is recommended according to manufacturer with TA.Cultivate 10 clones that obtained, DNA isolation and order-checking.Identified other 60 bases of 5 ' sequence in this way, it is corresponding to the base 1 to 60 of SEQ ID NO:45.

The feature of the aminoacid sequence of mice cDNA and prediction

Coding people α _dThe collating sequence of the mice cDNA that infers congener be shown in SEQ IDNO:45.Though the homology between the external structure territory of people and cloned mouse is very high, the homology in cytoplasmic structure territory only is 30%.Viewed difference may show the C-terminal function difference between people and the murine protein.The difference in cytoplasmic structure territory also may be caused by montage difference in addition, or shows other β ₂The existence of integrin gene.

At amino acid levels, by the measurable a kind of albumen of mice cDNA (SEQ ID NO:46), this albumen and mice CD11a have 28% homogeneity, with mice CD11b 53% homogeneity is arranged, with people CD11a 28% homogeneity is arranged, with people CD11b 55% homogeneity is arranged, have 59% mutually, with people α with people CD11c _d70% homogeneity is arranged.With people α _dIts mice congener of inferring of aminoacid sequence and same length in cytoplasmic structure territory compare, the result shows that these zones have identical length but different primary structures is arranged.These regional sequence lengths are similar to be shown and changes rather than the splice variant form between being kind.When with embodiment 20 above in prediction rat polypeptide find that mice and rat cytoplasmic structure territory show the homogeneity greater than 60% when comparing.

Embodiment 29

Separate other mice α _dThe cDNA clone is used for the sequence checking

Be the mice α that introduces among the checking embodiment 28 _dNucleic acid and aminoacid sequence, separate other mice sequence be used for the proof.

Use the mouse thymus oligomerization dT-primer cDNA library (Stratagene) among a mice spleen random primer library and the λ ZAP_II, with two kinds of homology α _dThe method of probe hybridization is carried out the separation of mice cDNA.The library is by about 5 * 10 ⁵The every 15cmLBM flat board of plaque carries out bed board.Plaque is offered (Amersham) on the Hybond_ nylon membrane, with film degeneration (0.5M NaOH/1.5 M NaCl), neutralization (0.5 M Tris alkali/1.5 M NaCl/11.6 HCl), washing (2 * SSC saline solution).Method with ultraviolet radiation is linked to DNA on the filter membrane.

Probe uses preparation in the primer PCR reaction of introducing below under the following conditions.Sample is 94 ℃ of incubations 4 minutes, carry out then 30 circulation temperature step orders (94 ℃ 15 seconds, 50 ℃ 30 seconds, 72 ℃ 1 minute, use Perkin-Elmer 9600 thermal cyclers.

3 ' probe be about 900 bases and cross over zone (the SEQ IDNO:1) from nucleotide 2752 to 3651 (5 ' →) 3 '), and prepare with the primer 11.b-1/2FOR11 and the 11.b-1/1REV1 that are shown in SEQ ID NO:69 and 74 respectively.This probe is used for first cover and offers.

5 ' probe is about 800 bases and crosses over zone (the SEQ IDNO:1) (5 ' → 3 ') from nucleotide 149 to 946, and prepares with the primer 11.b-1/2FOR1 and the 11.a-1/2REV2 that are shown in SEQ ID NO:50 and 85 respectively.This probe is used for second cover and offers.

In the 3rd cover was offered, two kinds of probes one introducing above were used from identical flat board.

Two kinds of probes that use is introduced above screen about 500,000 plaques, and probe carries out labelling by the same procedure of 20 kinds of introductions of embodiment.Label probe added in the prehybridization solution contain 45% Methanamide and at 50 ℃ be incubated overnight.Filter membrane is in room temperature (22 ℃) washed twice in 2 * SSC/0.1%SDS, and last washing is carried out in 2 * SSC/0.1%SDS at 50 ℃, on Kodak X-Omat AR film at-80 ℃ with intensifying screen radioactive automatic developing 19 hours.

At least 13 plaques that provide positive signal on the thing offering of duplicating are carried out second take turns screening, second takes turns screening is undertaken by the introduction of first screening, except following variation, 3 ' and 5 ' labeled primer all be used for hybridization, and add and once use 2 * SSC/0.1%SDS in addition 65 ℃ last washing.Carry out 2.5 hours radioactive automatic developing by top introduction.

Change 13 plaques (called after MS2P1 to MS2P13) that provide positive signal over to low Mg ⁺⁺In the phage diluent.Under described in the above the same terms, use can with annealed T3 of Bluescript_ phasmid and T7 primer among the ZAP_II (sequence is introduced in front), measure the size of inserting son with pcr amplification (Perkin-Elmer 9600 thermocycler).Stripe size in 500 bases to the scope of 4kb.Phasmid is reverse with M13 and M13.1 (being respectively SEQ ID NO:40 and 41) separated, prepared and check order.To 5 among 13 clones: MS2P-3, MS2P-6, MS2P9, MS2P-12, MS2P-13 check order, and they are together, represented from 5 ' first termination codon of about base 200 to the 3 ' end of end after the zone of about 300 bases.

Automatic sequencing is undertaken by the introduction among the embodiment 4.At first use M13 and M13 reverse.1 primer (being respectively SFQ ID NO:40 and 41) is measured each clone's end sequence, and determines its position with respect to construct #17 (SEQ ID NO:45).Each clone uses the primer suitable to the specific region (below listing in) to check order fully then.

11.b-1/2FOR1 5′-GCAGCCAGCTTCGGACAGAC-3′

(SEQ?ID?NO：50)

11.a-1/1FOR2 5′-CCGCCTGCCACTGGCGTGTGC-3′

(SEQ?ID?NO：60)

11.a-1/1FOR3 5′-CCCAGATGAAGGACTTCGTCAA-3′

(SEQ?ID?NO：61)

11.b-1/2FOR4 5′-GCTGGGATCATTCGCTATGC-3′

(SEQ?ID?NO：62)

11.b-1/2FOR5 5′-CAATGGATGGACCAGTTCTGG-3′

(SEQ?ID?NO：63)

11.b-1/2FOR6 5′-CAGATCGGCTCCTACTTTGG-3′

(SEQ?ID?NO：64)

11.b-1/2FOR7 5′-CATGGAGCCTCGAGACAGG-3′

(SEQ?ID?NO：65)

11.b-1/2FOR8 5′-CCACTGTCCTCGAAGCTGGAG-3′

(SEQ?ID?NO：66)

11.b-1/2FOR9 5′-CTTCGTCCTGTGCTGGCTGTGGGCTC-3′

(SEQ?ID?NO：67)

11.b-1/2FOR10 5′-CGCCTGGCATGTGAGGCTGAG-3′

(SEQ?ID?NO：68)

11.b-1/2FOR11 5′-CCGTGATCAGTAGGCAGGAAG-3′

(SEQ?ID?NO：69)

11.b-1/2FOR12 5′-GTCACAGAGGGAACCTCC-3′

(SEQ?ID?NO：70)

11.b-1/2FOR13 5′-GCTCCTGAGTGAGGCTGAAATCA-3′

(SEQ?ID?NO：71)

11.b-1/2FOR14 5′-GAGATGCTGGATCTACCATCTGC-3′

(SEQ?ID?NO：72)

11.b-1/2FOR15 5′-CTGAGCTGGGAGATTTTTATGG-3′

(SEQ?ID?NO：73)

11.b-1/2REV2 5′-GTGGATCAGCACTGAAATCTG-3′

(SEQ?ID?NO：74)

11.b-1/2REV3 5′-CGTTTGAAGAAGCCAAGCTTG-3′

(SEQ?ID?NO：75)

11.b-1/2REV4 5′-CACAGCGGAGGTGCAGGCAG-3′

(SEQ?ID?NO：76)

11.b-1/2REV5 5′-CTCACTGCTTGCGCTGGC-3′

(SEQ?ID?NO：77)

11.b-1/2REV6 5′-CGGTAAGATAGCTCTGCTGG-3′

(SEQ?ID?NO：78)

11.b-1/2REV7 5′-GAGCCCACAGCCAGCACAGG-3′

(SEQ?ID?NO：79)

11.b-1/2REV8 5′-GATCCAACGCCAGATCATACC-3′

(SEQ?ID?NO：80)

11.b-1/2REV9 5′-CACGGCCAGGTCCACCAGGC-3′

(SEQ?ID?NO：81)

11.b-1/2REV10 5′-CACGTCCCCTAGCAGTGTCAG-3′

(SEQ?ID?NO：82)

11.b-1/2REV11 5′-CCATGTCCACAGAACAGAGAG-3′

(SEQ?ID?NO：51)

11.b-1/2REV12 5′-TTGACGAAGTCCTTCATCTGGG-3′

(SEQ?ID?NO：83)

11.b-1/2REV13 5′-GAACTGCAAGCTGGAGCCCAG-3′

(SEQ?ID?NO：84)

11.a-1/1REV1 5′-CTGGATGCTGCGAAGTGCTAC-3′

(SEQ?ID?NO：85)

11.a-1/1REV2 5′-GCCTTGGAGCTGGACGATGGC-3′

(SEQ?ID?NO：86)

With the sequence editor, side by side, and with former isolating mice α _dSequence (construct #17, SEQ IDNO:45) compares.

New sequence alignment discloses, and 18 a bases deletion that starts from nucleotide 2308 is arranged in construct #17, and it is moving that this deletion does not cause reading to frame shift.Clone MS2P-9 by top introduction order-checking also shows identical 18 bases deletion.This deletion is found in 50% contain in this regional cloned mouse and take place, but at rat or people's α _dBut do not detect among the clone.The feature of 18 bases deletion is the palindrome AAGCAGGAGCTCCTGTGT (SEQ IDNO:91) of one 12 base.Inverted repeat in this nucleotide sequence is that self is complementary, and may form an outside ring, causes excision in the reverse transcription process.The mice α that contains these 18 extra bases _dSequence is listed in SEQ ID NO:52, and its deduced amino acid is listed in SEQ ID NO:53.

Embodiment 30

In situ hybridization in the mice

Then determine mice α _dTissue distribution, provide with embodiment 6 in the people α that introduces _dThe comparison that distributes.

Adopt external rna transcription and integration ³²The method of S-UTP (Amersham) has prepared the mRNA probe of the 200bp of a strand from a dna profiling, this probe is corresponding to the nucleotide 3460 to 3707 in mice cDNA cytoplasmic tail territory.

Use radiolabeled strand mRNA probe that the full embryo of mice (collecting in after fertilization 11-18 days) is comprised that with different mouse tissues spleen, kidney, liver, small intestinal and thymus carry out in situ hybridization.

It is thick that tissue is cut into 6um, and (Burlingame CA) on the microscope slide of bag quilt, is stored in-70 ℃ for Vector Laboratories, Inc to stick to Vectabond.Before use, microscope slide is taken out from-70 ℃, placed about 5 minutes at 50 ℃.To cut into slices in 4% paraformaldehyde 4 ℃ and fix 20 minutes, dewater at 4 ℃ with the ethanol gradient (75-95-100%) that improves constantly, each concentration 1 minute, at room temperature air drying is 30 minutes.To cut into slices in 70% Methanamide/2 * SSC 70 ℃ of degeneration 2 minutes, rinsing was 2 times in 2 * SSC, with the ethanol gradient dehydration of introducing above and air drying 30 minutes.Hybridization is containing 6 * 10 at 55 ℃ ⁵The cpm/ section ³⁵S labeling nucleic acid probe and in the water that pyrocarbonic acid diethyl ester (DEPC) is handled final concentration be spend the night in the solution of 50% Methanamide, 0.3M NaCl, 20mM Tris-HCl, pH7.5,10% dextran sulfate, 1X Denhardts solution, 100mM dithiothreitol, DTT (DTT) and 5mMEDTA (12-16 hour) carry out.After the hybridization, section was washed 1 hour in 4 * SSC/10mM DTT in room temperature, washed 40 minutes in 50% Methanamide/2 * SSC/10mM DTT at 60 ℃, washed in 2 * SSC 30 minutes in room temperature, washed in 0.1 * SSC 30 minutes in room temperature.The dehydration of will cutting into slices, air drying 2 hours, with Kodak NTB2 photoemulsion bag quilt, air drying 2 hours, colour developing (in dark fully after 4 ℃ of preservations), and with hematoxylin/Yihong negative staining.

The spleen tissue shows main strong signal at red pulp.This collection of illustrative plates is consistent with the distribution of tissue macrophages in the spleen, but does not get rid of other cell type.

Embodiment 31

The preparation of mice expression construct

For structure comprises the α with the people _dThe expression plasmid that shows the mice cDNA sequence of homology will be connected from insertion of clone A1160 and B3800.But before this connects, one section 5 ' targeting sequencing that contains an initial methionine is joined clone A1160.The primer of design called after " 5 ' PCR is leading " (SEQ ID NO:47), this primer contains: (1) is the identical non-specific base of being convenient to digest at position 1-6; (2) be convenient to the BamI site (underscore among the SEQ ID NO:47) of the position 7-12 of sub-clone in the expression vector; (3) position 13-18 total Kozak sequence; (4) contain the signal sequence of an initial methionine codon (runic among the SEQ ID NO:47); (5) overlapping with 5 ' sequence of clone A1160 specifically so that other 31 bases of primer annealing.Use second and be called the primer of " 3 ' terminal frag " (SEQ ID NO:48) and primer " 5 ' PCR is leading " to come from clone A1160 amplification to insert son.

5′-AGTTAC GGATCCGGCACCATGACCTTCGGCACTGTGATCCTCCTGTGTG-3′

(SEQ?ID?NO：47)

5′-GCTGGACGATGGCATCCAC-3′ (SEQ?ID?NO：48)

The PCR product that is obtained can not be digested by BamHI, and the base number deficiency that restriction site is preceding is described, has suppressed the identification of enzyme.The length of " tail " sequence before the BamHI site repeats PCR in the first time on the pcr amplification product in increase by 5 ' primer (SEQ ID NO:47).Designed a 5 ' primer that is called mAD.5 ' .2 (SEQ ID NO:49), it has extra non-specific base at position 1-4, and contains used with the front specifically eclipsed other 20 bases of " 5 ' PCR is leading " primer sequence.

5′-GTAGAGTTAC GGATCCGGCACCAT-3′ (SEQ?ID?NO：49)

It is the PCR reaction of template that primer " mAD.5 ' .2 " and " 3 ' end frag " are used for simultaneously with the product of amplification for the first time.Program sub-clone that second time of obtaining, the PCR product was recommended according to manufacturer in plasmid pCRtmII (Invitrogen), and is transformed in the One shot competent cell (Invitrogen).With BamHI and EcoRI restriction analysis a clone who contains the PCR product, and check order.Confirmed after the sequence that digestion separates insertion with gel-purified with EcoRI with BamHI.

Separates from clone B3800 with EcoRI and NotI digestion, gel-purified and to insert subly, and add in the segmental coupled reaction of A1160 BamHI/EcoRI that contains expansion.Being connected 14 ℃ carried out 14 hours.In coupled reaction, add carrier pcDNA.3 (Invitrogen) and extra ligase, continue reaction 12 hours with BamHI and NotI digestion.One five equilibrium thing reactant mixture is transformed in the competent escherichia coli cell, cultivate the clone who is obtained, use primer 11.b-1/2FOR1 and 11.b-1/2REV11 (being respectively SEQ ID NO:50 and 51) to come a positive colony is identified with pcr analysis.These primers have been crossed over A1160 and B3800 fragment, and therefore detecting amplified production just shows that two fragments connect.The sequence of positive colony verifies with the primer of listing in SEQ ID NO:50 and 51, and this can increase behind the initial methionine from base 100 to 1405 to primer.

Embodiment 32

A kind of structure of knock-out mice

For estimating the mice α that infers more accurately _dThe immunological role of cDNA encoded protein has designed a kind of " knocking out " mice, and in this mice, coding is inferred α _dThe genomic dna sequence of congener destroys with homologous recombination.Just can estimate by the proteic meaning of ruined gene code like this with the shortage of encoding proteins.The preparation that " knocks out " mice is introduced among the et al., Mol.Cell.Biol.13:2134-2140 (1993) at Deng.

Design such mice and contain the plasmid of preparing the gene that " knocks out " with the homologous recombination incident from structure.Use one the 750 base pair fragment (being equivalent to nucleotide 1985 to 2733 among the SEQ ID NO:45) of mice cDNA from a λ FIXII genomic library, to identify the mice α that coding is inferred _dThe mice genome sequence of congener.First screening has produced 14 positive colonies, and wherein 7 confirm with programmed screening.Obtain the liquid lysate from two plaques that provide peak signal, and separate λ DNA with traditional method.Restriction endonuclease map and Southern analytical proof clone's the correctness of one of them called after 14-1.Separate insertion DNA with NotI digestion.With this fragment cloning to Bluescript_SKII ⁺

For identifying a restricted fragment (it is said that this length is the probability maximum that makes the homologous recombination incident) that is about 9-14kb, carry out Southern hybridization with 750bp cDNA probe.Before hybridization, clone 14-1 is set up restriction endonuclease map.The fragment of a 12kb is accredited as possible material standed for, and with this fragment sub-clone to pBluescript_SKII ⁺In a position, the flank that makes mouse DNA is a thymidine kinase coding box.This clone be the analysis showed that further with a kind of I domain probe (being equivalent to nucleotide 454-1064 among the SEQ ID NO:45) this clone does not contain I domain coded sequence.

Use identical I domain probe, λ FIXII genomic library is screened once more.Beginning has detected 6 positive colonies, and one of them keeps positive when programmed screening.The DNA of clone and separate is strong with a kind of I domain probe reaction in Southern analyzes thus.But do not detect any reaction with original 750bp probe, this illustrates that this clone comprises 5 ' zone of the nucleotide 1985-2773 of SEQ ID NO:45.

In addition, can not may show that this clone is the another kind of member of integrin protein family with the 750bp probe hybridization.For determining whether this explanation correct, with the sub-sub-clone of the insertion of 13kb to pBluescript_SKII ⁺The DNA of the purification α that is equivalent among the SEQ ID NO:52 _dThe primer of I domain nucleotide sequence 441-461,591-612,717-739 and reverse 898-918 checks order.Only when using first 4441-4461 primer, obtained sequence information, and only have 5 of I domain '-the most exon effectively increased.The remainder of I domain is not amplified.Therefore, the clone who is obtained contains mice α _d5 of the exon 6 of gene and this exon ' and the intron sequences of 3 ' end.Exon 7 is not in the clone.After the order-checking, prepared a construct that contains neomycin resistance and thymidine kinase gene.

With neomycin resistance (neo ^r) gene is inserted in the plasmid that is obtained in the mode of the albumen coded sequence that interrupts genome mouse DNA.Therefore the plasmid that is obtained contains a neo in the mouse gene group DNA sequence ^rGene, all these is positioned at a thymus kinases coding region.The plasmid that needs to make up in this way makes homologous recombination be better than recombinating at random [Chisaka, etal., Nature 355:516-520 (1992)].

Another kind of strategy is used to preparation and is applicable to generation α _dThe construct of knock-out mice.Give Genome Systems with two cover oligonucleotide primers, (St.Louis MO) comes carrying out high strict pcr analysis by the big insertion library of embryonic stem cell genomic DNA preparation Inc..Primer is corresponding to α _dFirst and last exon of middle I domain.Identified three clones, wherein two (called after 1117 and 1118) can react with two kinds of primers, and another (called after 1119) can only use the primer amplification from last exon.

A mice genome α _dThe evaluation of DNA

From clone 1117 and 1118 bacterial lysate according to indication (GenomeSystems, Inc.) the preparation plasmid DNA of manufacturer.α _dInserting son uses oligonucleotide madk.f1 (SEQID NO:104) and madk.r1 (SEQ ID NO:105) and madk.r2 (SEQ ID NO:106) to verify with PCR.

madk.f1 SEQ?ID?NO：104

TGT?CCA?GGA?CAA?GAG?ATG?GAC?ATT?GC

madk.r1 SEQ?ID?NO：105

GAG?CTA?TTT?CAT?AGC?AAG?AAT?GGG

madk.r2 SEQ?ID?NO：106

TAT?AGC?ATA?GCA?AAT?GAT?CC

Two kinds of plasmids are divided into the five equilibrium thing to be digested with restriction restriction endonuclease BamHI, PstI, SacI, SalI, SmaI, XbaI and XhoI (Boehringer Mannheim).Each sample digestion is separated on 0.8% agarose gel, and polynucleotide are transferred to Hybond_-N ⁺(Amersham) analyzes on the nucleic acid transfer membrane.Trace is used ³²P random labelling primed DNA is that probe detects, ³²P random labelling primer prepares with the 1.6kb template, and this template is that primer produces with PCR with oligonucleotide madfor 1 (SEQ ID NO:107) and madrev 1 (SEQ ID NO:108).

madfor1 SEQ?ID?NO：107

ATG?GTC?CGT?GGA?GTT?GTG?ATC

madrev1 SEQ?ID?NO：108

TCG AGA TCC ACC AAA CTG CAD hybridization is spent the night at 42 ℃ in the SSPE that contains 50% Methanamide and is carried out.The trace of labelling in 2 * SSPE room temperature washing 5 times.Radiolabeled band is used trace was shown-70 ℃ of exposures on KodakX-Omat radioactive automatic developing film in 2 hours.

Two interested fragments have been identified from cloning 1118: the SacI fragment of a 4.1kb and the XbaI fragment of a 8.3kb.Clone 1118 SacI and whole sample contents of XbaI digestion are connected to carrier pBluescript KS without being further purified ⁺In, after the connection, the total overall reaction content is transformed in the calcium-competent cell of e. coli tg1/λ SmR bacterial strain.Separate the clone who produces, overnight incubation is duplicated single stranded DNA in containing the 200 μ l selection culture medium of helper virus M13K07.The supernatant 10 μ l point samples that to take from each hole then are to Hybond_-N ⁺On the transfer membrane, hybridize with same probe of introducing above and program.By 9 positive colony amplification culture, and with Wizard_Plus Miniprep dna purification system (Progema) isolated plasmid dna from each culture.Confirm to insert in the isolating plasmid existence and the size of son with restriction endonuclease digestion and PCR.

Use carrier primer T3 and T7 and corresponding to mice α _dThe oligonucleotide primers of sequence carries out sequencing to three clones.These three clones and mice cDNA comparison shows that with the sequence that Geneworks software carries out all these three clones contain mice α simultaneously _dExons 1 and exon 2.Being called as the Changke of A grand is the long XbaI clone of a 8280kb.Two short clones are called as E and H in addition, and they are identical long SacI clones of 4112kb.

Select the 8280kb clone further to develop.

Embodiment 33

Rabbit α _dThe clone

The structure and the screening in rabbit cDNA library

People α in rat and the mice _dThe evaluation of congener has promoted the research to the rabbit congener, and the rabbit congener will be very useful in the rabbit model of the human diseases of above introducing.

Use Invitrogen FastTrack test kit (San Diego, CA) and the reagent that provides of the test kit program of recommending according to manufacturer from a full rabbit spleen, prepare Poly A ⁺RNA.From 1.65 gram tissues, separated the Poly A of 73 μ g ⁺RNA.Use one from Stratagene (La Jolla, test kit CA) come to make up a ZAP_ with rabbit spleen RNA and express the cDNA library.The cDNA that obtains directly is cloned into the EcoRI and the XhoI site of the λ arm of pBK-CMV phasmid carrier.With Gigapack_II Gold (Stratagene) the λ arm is packaged in the phage particle.The titre in the library that is obtained is estimated as 8 * 10 ⁵Granule, average insertion size is 1.2kb.

Grow once the collecting cell lysate in flakes with amplified library by the bed board plaque.Bed board is carried out by the every 150mm escherichia coli of about 30,000 plaque-forming units (pfu) flat board in library after the amplification, plaque is formed the mixture that produces.Phage DNA is transferred to Hybond_N ⁺On the nylon membrane (Amersham, ArlingtonHeights, Illinois).Film is with two kinds of radiolabeled mice α of random primer _dThe mixture of PCR dna probe is hybridized.First probe prepares with a PCR product of crossing over the nucleotide 149-946 of SEQ ID NO:52.Second probe is from the PCR product of the nucleotide 2752-3651 that crosses over SEQ ID NO:52.Probe carries out labelling with random primering (BoehringerMannheim random primer dna marker test kit), and reactant mixture removes uncorporated nucleotide by a Sephadex_G-50 post.Hybridization solution is by 5 * SSPE, 5 * Denhardts, 1%SDS, 40% Methanamide and 1 * 10 ⁶The dpm/ml label probe is formed.Hybridization was carried out 16-18 hour at 42 ℃.Filter membrane thorough washing at room temperature in 2 * SSPE/0.1%SDS, and exposure shows the plaque of any hybridization on x-ray film.

Two obvious and people α have been identified _dThe clone of sequence homology.Clone #2 be about 800bp, and with people α _d5 ' terminal matching.Clone #2 comprises an initial methionine and complete targeting sequencing.Clone #7 is about 1.5kb and comprises an initial methionine.5 of clone #7 ' is terminal terminal overlapping with 5 of clone #2 ', and the site of 3 ' end sequence after I domain sequence stops.With primer walking method clone #7 is carried out complete order-checking.Nucleotide and the deduced amino acid of clone #7 are listed in SEQ ID NO:100 and 101 respectively.

By clone #2 and the definite rabbit α of clone #7 _dPrediction N-terminal aminoacid sequence show this albumen and people α _d73% homogeneity is arranged, with mice α _d65% homogeneity is arranged, 58% homogeneity is arranged with mice CD11b, people CD11b and people CD11c.The nucleotide sequence of clone #2 is listed in SEQ ID NO:92, and the aminoacid sequence of its prediction is listed in SEQ ID NO:93.

The rabbit of usage flag is cloned #7 and screens once more obtaining this cDNA fragment library, manages to separate the rabbit α of total length _dCDNA.Identified 25 other clones, one of them is called clone 49 the maximum that is confirmed as.Carry out complete order-checking with the nested type deleting technique to cloning 49.Clone 49 nucleotide and aminoacid sequence are listed in SEQ ID No:102 and 103 respectively.Because clone #7 and #49 are not overlapping, design oligonucleotides separates the sequence of losing as the primer among the PCR that uses the first chain rabbit spleen cDNA.

The relation of these two part clones' the derivation aminoacid sequence and the aminoacid sequence of other leukocyte integrins is illustrated in table 1.

Table 1

β ₂The integrin family member is at the homogeneity percentage ratio of amino acid levels

People α _dRabbit #7 rabbit #49

People α _d100 74 80

Mice α _d70 67 74

Rat α _d70 66 73

Mice CD11a is * 28 28 at random

Mice CD11b 55 59 53

People CD11a 36 28 28

People CD11b 60 58 55

People CD11c 66 59 62

If *＜25% homogeneity is only for comparison at random, nonsensical.

Separated rabbit α _dThe clone just can express this albumen, both can be expressed in the surface of transfectional cell, also can express with the total length or the clipped form of solubility.Then this albumen is prepared monoclonal antibody originally as immunity, be used for the rabbit model of human disease's state.

Embodiment 34

Monkey α _dSeparation

The preparation affinity column

A kind ofly can from the monkey spleen, separate α for preparing _dAffinity column, will resist people α _dEach 10mg of antibody 212D and 217L is to containing 0.1M NaHCO ₃, 0.5M NaCl, pH8.3 the coupling buffer dialysed overnight.The CNBr Sepharose_4B (Pharmacia, Piscataway NJ) of the about 1.0g of program preparation that recommends according to manufacturer mixes the 1.0mL resin with the antibody of every kind of dialysis.The slurry that is produced spends the night 4 ℃ of rotations and mixes.In the Beckman desk centrifuge centrifugal 5 minutes, obtain link coupled resin at 1000rpm.Collect the not supernatant part of absorption, analyze the not existence of coupling protein with spectrophotometer.The result shows that all available antibody all combine with gel-type vehicle.Room temperature sealing 2 hours, resin washed through a series of high and low pH changes in coupling buffer not link coupled active group on the resin, then with containing 0.1M NaC with the 1M ethanolamine ₂H ₃3H ₂The acetate buffer of O and 0.5M NaCl, pH4.0 washs at last.All resins all are stored in 4 ℃ in coupling buffer.

The preparation of monkey spleen

Female macaque spleen is available from the Regional Primate Center of University of Washington.With collagenase D (Sigma) the injection spleen tissue of 100U/ml, and be cut into small pieces.What then tissue is suspended in small size contains 50mM Tris, 150mM NaCl, 2mM CaCl ₂, 2mM MgCl ₂, pH8.0 and 1.0%Triton X-100_ lysis buffer, and be stored in-70 ℃.Add protease inhibitor PLA (pepstatin A, leupeptin and press down the mixture of enzyme peptide, all from Sigma) and 4-(2-amino-ethyl)-benzene sulfonyl fluorine hydrochloric acid (AEBSF) (NovaBiochem) to prevent the proteolysis in the storage process.Storage tissue is up to obtaining 6 macaque spleens.

The spleen of 6 monkeys is mixed, in a Waring agitator, containing 25mM Tris, 0.15M NaCl, 002%NaN ₃, 1.0%Triton_, 1X PLA and 0.1mM AEBSF the TSA lysis buffer in homogenate, carry out three circulations altogether, each 10 seconds.Collect lysate, and on a rotation platform 4 ℃ placed 1 hour.In the Beckman desk centrifuge centrifugal 15 minutes then with 3000rpm.Collect supernatant, and discard sedimentary cell residue.Having collected cumulative volume altogether is the lysate of 550ml, with lysate 4 ℃ with in the 1M ethanolamine, handle in advance with the CNBr Sepharose_ incubation in capping site 2 hours.Behind the incubation, the centrifugal resin of removing is collected supernatant.

Affinity purification and order-checking

With the spleen lysate of preparation as stated above in two, the CNBr Sepharose_ gel with 212D and 217L preparation mixes respectively.The slurry that produces was mixed 3 days 4 ℃ of rotations, on the Beckman desk centrifuge, collected the not part of absorption in centrifugal 10 minutes then, and preserve with 1500rpm.Gel is transferred in the 15ml centrifuge tube order washing in the D-PBS of several volumes.The five equilibrium thing of 100 μ l of every kind of gel is boiled rapidly in the reduced form sample buffer that contains 0.1M Tris-HCl, pH6.8,2%SDS, 20% glycerol and 0.002% bromophenol blue, 10% beta-mercaptoethanol (final concentration 5%), and last 6.0% polyacrylamide sds gel (SDS-PAGE), analyzing proteins.Gel dyes with coomassie, detects to follow the proteic and α of a large amount of backgrounds _dThe albumen consistent with the CD18 molecular weight.

For raising has and α _dThe proteic purification of similar molecular weight washs every kind of two 100 branch things such as μ l that combine proteic gel with diverse ways.In one approach, gel washs in the buffer that contains 150mM NaCl, 10mM Tris, 1.0%Triton-X100_, pH8.0 for several times.In the second approach, gel washs with same procedure, but bonded albumen carries out eluting with 0.05M glycine, pH2.4 in washing the last time.As before, eluted protein boils rapidly in the reduced form sample buffer, and analyzes on 6.0%SDS-PAGE.From the resin that washs low pH glycine buffer, coomassie dyeing only detects and α _dTherefore the albumen consistent with CD18 select this method.Be the albumen that separation is used to check order, remaining CNBr Sepharose_ resin is by the introduction of front washing four times, and the resin with about 3/4ths is suspended in the 0.05M glycine, pH2.4 of 2ml, acutely shakes up.Make resin precipitated in centrifugal 3 minutes, and collect the not part of absorption.Then with gel in glycine buffer again washing once this washings is mixed with the not bound fraction of front.With this mixing portion 4 ℃ to the D-PBS dialysed overnight, liquid is changed twice in the centre.After the dialysis, make volume be reduced to 1ml sample drying.

For checking order, with eluate on 7.0% separation gel separately, the method for introducing by embodiment 2 with albumen transfer to Immobilon (PVDF) film (Millipore Bedford, MA) on.In brief, gel is washed once in deionized water, and in containing the 10mM cyclohexylamine of 10% methanol-propane sulfonic acid pH of buffer 10.5 (CAPS) balance 15 to 45 minutes.The PDVF film is washed in methanol and deionized water, then balance 15 to 30 minutes in the CAPS transfering buffering liquid.Albumen was transferred on the PDVF film 70 volts of transfers in 3 hours, then it was dyeed 10 minutes in the filtering 0.1%R250 coomassie dyestuff of process.Film washs decolouring three times in 50% methanol/10% acetic acid, washed 10 minutes at every turn, in filtered water, wash once again, and dry.

All detect the major protein band of about 150kD and 95kD from 212D and the link coupled resin of 217L, these two protein bands are consistent with front detected albumen on the AG gel.Observing a band that definition is poor slightly that is positioned near below the 150kD albumen on the film from 217L coupling resin, but after the film drying, detecting less than this band.Downcut the 150kd band from every film, (Foster City, CA) 473 type protein sequencing instrument directly check order according to the program of manufacturer's recommendation with Applied Biosystems.

The result shows, has the aminoacid sequence of listing in SEQ ID NO:109 with the proteic amino terminal sequence of monkey of 212D coupling resin isolation.And have the sequence of listing in SEQ ID NO:110 with the proteic aminoterminal of 217L coupling resin isolation.Undetermined residue of " X " expression among the SEQ ID NO:110.

212D-coupling protein SEQ ID NO:109

NLDVEEPTIFQEDA

217L coupling protein SEQ ID NO:110

NLDVEEPTIFXEDA

Monkey sequence and people α _dAnd β ₂In the integrin family other α chains amino terminal sequence relatively be shown in table 2

Table 2

Monkey α _dAmino terminal sequence and people β ₂The comparison of beta 2 integrin alpha subunit

Protein S EQ ID NO: amino terminal sequence

Monkey α _d111 F N L D V E E P T I F Q E D A

People α _d112 F N L D V E E P T I F Q E D A G G

People CD11c 113 F N L D T E E L T A F V D S A G

People CD11b 114 F N L D T E N A M T F Q E N A R G

Based on sequence homogeneity, conclusion is that 212D and 217L can both discern monkey and people's α _d

Embodiment 35

The antigenic feature analysis of 217L

Based among the embodiment of front from the monkey spleen sedimentary proteic N-terminal sequence, can reach a conclusion, 212D and 217L can discern monkey and people's α _dBut immunocytochemistry (ICC) is analyzed and immunoprecipitation experiment shows that 217L has the extra response characteristic that 212D does not have.Use is tested the cross reactivity that detects 217L and CD11c (the most closely-related leukocyte beta 2 integrin alpha chain) and CD11b at the FACS and the ICC of the antibody of all α chains.Therefore, possible 217L also discerns α _dConformation, glycosylation or splice variant or and α _dThe new α chain that sequence homology is arranged.

The antigen of antibody 217L identification has unique distribution in the sarcoidosis lung tissue (seeing embodiment 18), and not overlapping with the dyeing collection of illustrative plates of CD11c, this shows that this antigen may have biological significance.Therefore, for further understanding the antigenic meaning of 217L, need analyze this albumen and this proteic DNA of coding, and adopt different approach to carry out.

Use antibody 217L to come a kind of albumen composition of immunoprecipitation from people's dendritic cell or peripheral blood, then sedimentary albumen is carried out the N-terminal order-checking.Sequence analysis will disclose, the albumen of on peripheral blood cells, discerning whether with α _dHas aminoterminal homogeneity.To handle with deglycosylating enzyme from dendritic cell or the sedimentary albumen of peripheral blood cells then, and with originate from other sedimentary CD11c and α _dCompare, the molecular weight ratio that the one-level aminoacid sequence is provided.

In addition, use whole α _dCDNA carries out probe hybridization to the cDNA library that is prepared by dendritic cell RNA under low stringency condition.The reaction clone uses whole clone's length nucleic acid sequencing is analyzed, and determines whether there is non-α among the clone _dSequence.

Embodiment 36

Measure α _dThe animal model of therapeutic use

Immunohistology data in the dog and the in situ hybridization in rat and the mice are definite, in spleen, and α _dMainly express α by the macrophage in red pulp and the lymph node _dIn medullary cord and hole, be found.Its expression map is very similar to two kinds of antigenic expression maps of Mus having reported, determine with two kinds of monoclonal antibody F4/80 and SK39.And the antigenic biochemical character of these mices is verified, and they are different from α _d, α _dDetermined probably and mice antigen F4/80 and the determined identical macrophage subgroup of SK39.

In mice, SK-39 is positive, and macrophage is differentiated in splenic red pulp and lymph bend hawsers, and in splenic red pulp, they may participate in removing allogenic material from circulation.[Jutila，et?al.，JLeukocyte?Biol.54：30-39(1993)]。The positive macrophage of SK-39 also is in the news and is positioned at the position of acute and chronic inflammation.And the mononuclear cell that accumulates in the peritoneal cavity that TGA brings out inflammation is also expressed SK39 antigen.In summary, these discoveries show, if SK39 ⁺Cell also is α _d ⁺, these cells will be responsible for removing allogenic material in spleen so, and participate in the inflammation that macrophage plays an important role.

Though α _dFunction not clear, The Characteristics gets clearer other β ₂Integrin has been shown participation and has adhered to incident very widely, comprises promoting cell migration, reinforcement to engulf, promote the incident of cell-cell interaction, and all incidents that cause inflammatory process to raise.Therefore, very possible, intervene normal α _dFunction also can be intervened the inflammation that macrophage plays an important role therein.Such anti-inflammatory effect is produced by following factors: i) stop macrophage to be assembled in the inflammation site, ii) stop macrophage to activate or iii) disturb the effector function of macrophage in the inflammation site, these effector functions are by specific autoimmune response or as the onlooker of cell injury and injuring normal cell.

Evidence suggests that morbid state that macrophage plays an important role comprises the diabetes and the enteritis disease of multiple sclerosis, arthritis, transplanting atherosclerosis, some type in lysis.Animal model discussed below has shown many aspects of these human diseasess of reproducible.α _dWhether the function of inhibitor is tested in these model systems, exist with the probability of determining the corresponding human diseases of treatment.

Transplant atherosclerosis

Heart transplantation be at present some type of therapeutic intervention late period heart disease a kind of can be by being subjected to form.Owing to use cyclosporin A to make an annual survival rate bring up to 80%, main causes of death after producing gradual transplanting atherosclerosis and having become heart transplantation and survived a year.Nearest discovers, heart transplantation after 3 years tangible atherosclerotic occurrence scope be 36-44%[Adams, et al., Transplantation 53:1115-1119 (1992); Adams, et al., Transplantation 56:794-799 (1993)].

Transplant atherosclerosis and generally comprise diffusivity, occlusive, the interior membrane damage that can influence whole coronary vasodilator wall, and be accompanied by lipidosis usually.Though it is still unclear to transplant atherosclerotic pathology, the histocompatibility difference with donor and receptor is relevant probably for it, and is immunity in itself.From the histology, the interior diaphragm area of thickening mainly is that macrophage is formed, though also can see the T cell once in a while.Therefore express alpha probably _dMacrophage transplant atherosclerotic induce and/or develop in play an important role.In this case, α _dThe monoclonal antibody of function or micromolecular inhibitor (for example, the ICAM-R of solubility) can be for having accepted heart transplantation and having had the individuality that produces transplanting atherosclerosis risk that prevention is provided.

Though the atherosclerosis in the heart transplantation has caused biggest threat to life, transplant atherosclerosis and also see other solid organ transplantation, comprise kidney and liver.α _dThe therapeutic of blocking-up reagent is used the atherosclerosis that can prevent in other organ transplantations, reduces the complication that graft failure produces.

The atherosclerotic model of rat implantation relates to the allosome cardiac allograft of crossing minimum histocompatibility obstacle.When Lewis cardiac allograft thing was transplanted to the F344 receptor of MHC I and II type coupling, 80% alloplast survived for 3 weeks at least, and 25% graft is infinitely survived.In this rudimentary transplant rejection process, in donor's heart, form atherosclerosis.Tremulous pulse infringement in 120 days the alloplast generally has diffuse fibrous intimal thickening, and is as broad as long with the performance of the transplant graft atherosclersis infringement of finding in the human heart alloplast that repels.

Rat is transplanted with the unmatched heart of minimum histocompatibility antigen, for example Lewis to F-344.Regularly give rat α to transplant recipient _dSpecial monoclonal antibody or α _dMicromolecular inhibitor.Processing be hoped to reduce in non-repulsion donor's heart transplant graft atherosclersis generation.Use α _dMonoclonal antibody or micromolecular inhibitor are handled rat can be not limited to preventative processing.Blocking-up α _dFunction also can wish to reduce macrophage-mediated inflammation and the infringement of graft medium-sized artery is replied.

Cholesterol is raised the atherosclerosis in the rabbit

Add the atherogenicity food of thing and raise interior membrane damage [Rosenfeld, et al., the Arteriosclerosis 7:9-23 (1987) that the rabbit in about 12-16 week can produce the surface, chamber of the most of aorta ascendens of covering with a kind of cholesterol that contains; Rosenfeld, et al., Arteriosclerosis 7:24-34 (1987)].The atherosclerotic lesions of in these rabbits, seeing in the people, see similar.Contain a large amount of T cells in the infringement, the most CD45RO that express of these T cells, this is a kind of labelling relevant with memory T cell.The infiltrating T cell of half is also expressed MHC II class antigen approximately, and has some to express the IL-2 receptor, and this illustrates that these cells are in activated state mostly.

An aspect of atherosclerotic lesions finds in the rabbit that cholesterol is raised, but obviously do not have in rodent model, and the infringement that Here it is is rich in foam cell is gathered.Foam cell type macrophage it is believed that it is that low density lipoprotein, LDL (LDL) by specific receptor has absorbed oxidation causes.The LDL granule of oxidation is found some cell type is comprised that endotheliocyte and smooth muscle cell are deleterious.The LDL granule of possible deleterious, the oxidation of macrophage picked-up orders about macrophage activation, thereby the relevant inflammation of atherosclerotic lesions is worked as a kind of excimer.

To rabbit α _dPrepared a kind of monoclonal antibody, and handled the rabbit that cholesterol is raised with it.Processing comprises the preventative α of giving _dMonoclonal antibody or micromolecular inhibitor prove α _d ⁺The process of macrophage involved in diseases.Research in addition will prove at α _dMonoclonal antibody or micromolecular inhibitor can use in the rabbit that a kind of atherogenicity food raises detected vascular lesion to reply.

Insulin dependent diabetes mellitus (IDDM)

BioBreeding rat can spontaneous generation insulin dependent diabetes mellitus (IDDM) in the time of 70-150 in age days Mus.Use immunohistochemistry technique, can detect MHC II in early days in disease ⁺Class ED1 ⁺Macrophage is to the infiltration of islets of langerhans.Most macrophages show as and participate in cell residue or Normocellular engulfing.When disease progression, more macrophage is found and soaks in the islets of langerhans, accumulates in this site [Hanenberg, et al., Diabetologia 32:126-134 (1989)] though have a considerable amount of T cells and B cell afterwards also to be found.

As if the development of diabetes depends on early stage macrophages infiltration and T cell aggregation subsequently in the BioBreeding rat.With blocking of the infiltration of early stage macrophage effectively to islets of langerhans to the deleterious silica dioxide granule treatments B of macrophage B rat.When not had early stage macrophages infiltration, the histologic lesion of following that is caused by the lymphocyte populations autoaggression just can not take place.Monoclonal antibody OX-19 (rat CD5 is special) or monoclonal antibody OX-8 (CD8 is special) with the relevant disease phase of energy blocking t cell carry out the development that administration also can suppress diabetes effectively.

The central role that macrophage is risen in the pathology of this model makes and tests α with it _dThe inhibitor of function is very attractive.The rat that produces insulin dependent diabetes mellitus (IDDM) in advance surely in the heredity is used at α _dMonoclonal antibody or micromolecular inhibitor handle, and estimate advancing of disease.Prevent from or postpone clinical symptoms to begin just to prove α _dIn the infringement of macrophage, play a crucial role to islet cells.

Inflammatory bowel (clone disease, ulcerative colitis)

The animal model that is used for studying inflammatory bowel (IBD) is generally induced with deleterious stimulus object (for example acetic acid or trinitro-benzene-sulfonic acid/ethanol) per rectum administration.The inductive colitis of these reagent is the result of chemistry or metabolism damage, lacks the chronic or spontaneous recurrence inflammation relevant with people LBD.But the injection of a kind of use subserosa of nearest introduction is by the animal model of Peptidoglycan-polysaccharide (PG-PS) polymer of A type or D type streptococcus purification, seemingly more relevant model [Yamada with people LBD physiology, et al., Gastroenterology 104:759-771 (1993)].

In this model, PG-PS is expelled to the subserosa of colon far away.The inflammatory response that is produced divides two periods, is the acute attack of injection after three days at first, then the idiopathic chronic phase after 3 to 4 weeks.It is granulomatous in itself that later stage replys, and causes that colon thickens, adhesion, colon joint and mucosa injury.Except mucosa injury, PG-PS colitis often causes arthritis, anemia and the internal bud hepatitis that swells.The outer symptom of the intestinal of this disease makes this model can be used for studying clone disease, because much suffer from the torment that the patient of activeness clone disease is subjected to joint disease and hepatocholangeitis disease.

The granuloma infringement is the result of chronic inflammatory disease, and it is the gathering of cell and activation subsequently that chronic inflammatory disease causes monocyte/macrophage.In clone disease and above-mentioned animal model, there is the granuloma infringement, makes it become α _dMonoclonal antibody or α _dThe attractive clinical target of other inhibitor of function.α _dThe inhibitor of function is hoped to stop the formation of the infringement relevant with IBD or even reverses histologic lesion seen in the disease.

Arthritis

Seemingly a kind of multifactorial lysis of arthritis relates to multiple inflammatory cell type and comprises neutrophilic granulocyte, T lymphocyte and engulfing property macrophage.Though there has been multiple arthritis model, streptococcus whole cell peptidoglycan prepared product can produce the disease the most similar to human diseases.

In rat, the streptococcus cell wall is induced the periphery arthritis, it is characterized in that the disease process that shows effect repeatedly and alleviates subsequently, and finally cause destruction of joint [Cromartie, et al., J.Exp.Med.146:1585-1602 (1977) behind some months; Schwab et al., Infection and Immunity 59:4436-4442 (1991)].In the chronic phase of disease, the phagocyte of monokaryon or macrophage it is believed that in the destruction of synovial membrane and play a major role.And, the reagent that macrophage accumulates in the synovial membrane can be suppressed and inflammation and arthritic pathological characters can be reduced effectively.

Macrophage plays the role of a nucleus in causing arthritic synovial membrane destruction and is indicating, at α _dMonoclonal antibody or α _dThe inhibitor of function has the treatment potentiality in this disease of treatment.Other models as introducing in front prophylactically give α _dMonoclonal antibody or micromolecular inhibitor are to be hoped to stop or ameliorate osteoarthritis disease and prevent that synovial membrane from destroying.Intervene α _dThe reagent of function also can alleviate ongoing inflammation by stoping extra macrophage to gather the joint or blocking macrophage activation.Final result is to reverse ongoing destruction of joint and promote tissue repair.

Multiple sclerosis

Though the pathology reason of multiple sclerosis (MS) is not clear, think all that generally this disease is by discerning the autoantigen among the central nervous system and exciting the CD4 of inflammation cascade reaction ⁺T is cell-mediated.The immunne response that is produced causes extra inflammatory cell to comprise gathering to the effective activated macrophage of this disease.Experimental autoimmune encephalomyelitis (EAE) is a kind of animal model that can duplicate some aspect of MS.Recently, can show and can block clinical and histological disease with the monoclonal antibody [Huitinga, et al., Eur.J.Immuslol.23:709-715 (1993)] of the CD11b/CD18 reaction that exists on the struvite macrophage.These results show, α _dMonoclonal antibody or micromolecular inhibitor may be in the inflammatory response of blocking-up EAE effectively.Such reagent also has important therapeutic to use in the processing of MS.

The immune complex alveolitis

The pulmonary alveolar macrophage that is positioned at alveolar duct, trachea, connective tissue and the pleura gap of pulmonary is being represented the first line of defence of the surrounding material of lung opposing suction.Respond to material and comprise LPS, the IFN-γ of bacterial origin and the stimulation of immune complex, pulmonary alveolar macrophage discharges multiple strong inflammatory mediator, comprises the oxygen-derived free radicals and the nitrogen intermedium of high response.Though superoxide anion, hydrogen peroxide and nitric oxide (NO ^-) in removing pathogen, important function is arranged, but these materials can produce injury to organizing normally also.

In the rat model of an immune complex alveolitis, from the NO of pulmonary alveolar macrophage release ^-Shown and can mediate various pulmonary [Mulligan, et al., Proc.Natl.Acad.Sci. (USA) 88:638-6342 (1991)].NO ^-Also comprise that in the damage of other immune complexs mediation (Mulligan etc. are confirmed to be medium in above), and for example may work in the glomerulonephritis in disease cutaneous vasculitis.

NO ^-The tissue injury of mediation is not limited to the inflammation that immune complex participates in.For example, can be killed less the prominent neural NO that loses the cell plastid level by the nervelet erosion cell plastid generation of reagent such as PMA, LPS or IFN-γ stimulation ^-[Merrill, et aL, ImmunoL 151:2132 (1993)].Islet cells also is found NO ^-Sensitivity, and macrophage discharges the tissue injury [Kroncke, et aL, BBRC 175:752-758 (1991)] that this medium has been considered to participate in causing diabetes.In the time of more recently, final certification NO ^-Be released in the endotoxin shock work [MacMicking, et al., Cell 81:641-650 (1995)].When being given lipopolysaccharide (LPS), the normal wild type mice produces serious, progressive aortic pressure reduction, causes death.When responding to LPS, produce not too serious arterial pressure reduction but lack inductive nitric oxide production mice, and all survivals in processing.

In vitro tests shows, blocking-up α _dCan block macrophage (or common express alpha effectively _dLeukocyte) activatory some aspect, as NO ^-Discharge.There is anti-α in the pulmonary alveolar macrophage that stimulates with IFN-γ _dPolyclonal antiserum is (in rabbit at a kind of α _dThe preparation of I domain polypeptide) is found than the macrophage of handling with the contrast antiserum time and produces the NO that obviously lacks ^-Catabolite--nitrite/nitrate.This finds explanation, at α _dParticularly at α _dThe monoclonal antibody of I domain may be a kind of potential anti-inflammatory agent, and potential application is arranged in MS, diabetes, pneumonia and endotoxin shock.And, relative with the CD18 of the multiple leukocyte type of functionality of influence, α _dLimited distribution make it become target than CD18 more attractive, stop macrophage (or express alpha usually _dLeukocyte) activation.

The inductive alveolitis of rat IgG immune complex be widely used, understand the important experimental model of acute lung injury.This damage by through tracheal intubation to pulmonary inject anti-bovine serum albumin (BSA) antibody and then intravenous injection BSA induce.The immune complex that pulmonary's tracheole forms causes complement activation and neutrophil accumulation to pulmonary.Probably, then be that leukocyte exosmoses from blood and then passes lung epithelial after the formation of pulmonary's immune complex.Release medium comprises free radical, TNF-α and nitric oxide (NO subsequently ^-) from activated endothelial cells, neutrophilic granulocyte and the macrophage of involved in diseases process, discharge.The pathological characters of disease comprises that the vascular permeability that causes edema and alveolar gap to have a large amount of erythrocyte and PMNs increases.

In an inductive alveolitis rat model of immune complex at α _dThe special polyclonal antiserum of I domain detects.Anti-α _dPolyclonal antiserum and anti-BSA import in the lung through tracheal intubation at one time.Then pulmonary lesion gives BSA and a spot of by vein ¹²⁵The BSA of I labelling (about 800,000cpm) to induce, a kind of material in back is used for the edema that the quantitative measurement injury of lung produces.Pulmonary lesion was carried out 4 hours, and damage is estimated with the lung permeance value, and the lung permeance value is defined as pulmonary ¹²⁵The labelled amount that exists in the BSA of I labelling and 1.0ml blood ratio relatively.In general, the scope of the permeance value of positive control ratio is between 0.6 to 0.8, and the permeance value scope that negative control (not accepting the rat of BSA) has is 0.1-0.2.

Preliminary study shows, uses anti-α _dPolyclonal antiserum is handled the lung permeance value that can reduce greater than 50%, and this shows the alleviation greatly of injury of lung.In the past, can reduce by 60% permeance value with anti-CD18 processing.These discoveries show α _dMay be most important β in the acute lung injury process ₂Integrin, but can not accurately determine the effect that antiserum stops leukocyte to ooze out or pass lung epithelial from blood.

As α _dAlleviate the other evidence of injury of lung, the TNF-alpha levels in the bronchovesicular sucking-off liquid is estimated.Use anti-α _dAntiserum is handled to be found and can be made the TNF-alpha levels reduce about 4 times.TNF-α is regarded as the important medium in the acute pneumonia already, and causes inflammatory cell to gather inflammation part, cell-stimulating and tissue injury.Probably, anti-α _dAntiserum stops immobilized pulmonary alveolar macrophage activation in the forming process of immune complex alveolitis, thereby alleviates TNF-α and NO ^-Release, and reduce tissue injury and the neutrophil accumulation cause by these reagent subsequently.

The leukemic F344 rat model of LGL

The LGL leukemia of F344 rat is introduced as the transplantability leukemia with stable NK cytoactive to mid-term in early days as far back as 1980's.This leukemia is considered to can be used as model [Ward andReynolds, the Ain.J.Pathol.111:1-10 (1982) of people T γ lymphoma and T cell chronic lymphocytic leukemia; Stromberg, Am.J.Pathol.119:517-519 (1985); Reynolds, et al.J.Immunol.132:534-540 (1984)].This model can provide competent cell for studying LGL and NK cell function.The most interesting is that detected by facs analysis with the hamster murine antibody 205C of the Chinese People's Anti-Japanese Military and Political College as what introduce among the embodiment 26, there is α in these cell surfaces _dBecause should find, to α _dDetect in external (for example, with lysis test of introducing previously) and intravital effect.

The leukemic pathological characters of LGL comprises the petechial hemorrhage in liver, periphery lymphadenovaris and lung, brain and the lymph node of serious splenomegaly, white dot.Because α _dThe red pulp (on the splenic macrophage) that is present in the normal rat spleen, and the leukemic sign of LGL is serious splenomegaly, therefore can suppose α _dMale NK tumor cell also may " returning nest " or is limited to a kind of part that waits to determine, and in this hypertrophy.For checking this hypothesis, with the tumor cell radioactive label, and with or not with α _dAntibody treatment is expelled in the receptor rat together.Take out the spleen of these animals after three hours, detect the existence of NK tumor cell.Method and the result of experiment used of place of matchmakers more completely below.

Testing preceding 2 to 4 weeks at every turn, will transfer in the receptor rat available from the spleen of LGL leukemia rat and by the adopting property of tumor cell of following introduction preparation.Know that from the Histological research and the facs analysis of front the quick hypertrophy of tumor and the splenomegaly of generation occur in about 3 to 4 weeks after the transfer of adopting property.In first experiment, from an animal of accepting 4 weeks of tumor cell, take out spleen.With pincers spleen is cut into small pieces, and with small pieces existing under the condition of D-PBS by a mesh sieve, be prepared into a kind of single cell suspension.Cell suspension is collected in the 50ml centrifuge tube, and in the Beckman desk centrifuge at room temperature with 1500rpm centrifugal 10 minutes.Abandoning supernatant, cell suspend in 30ml D-PBS again.This cell suspension of about 5.0ml is laid on the Histopaque of 5.0ml, and with gradient at 1500rpm centrifugal 30 minutes.Collecting cell layer from these gradients merges, and counts with blood cell calculator.Cell quantity is adjusted to each receptor rat accepts 1.0 * 10 ⁷Cell, and have more some a little to offset the loss cell in washing and the preparation injection process.With cell suspension in NK " test media " (added antibiotic RPMI-1640, added 2%FBS), and with 10mCi's ⁵¹Chromium was 37 ℃ of labellings 1 hour.Behind the incubation, the volume of cell suspension is increased to 50ml with test media, and at centrifugal 10 minutes sedimentation cells of 1200rpm.Abandoning supernatant, cell is by introducing washed twice again.The cell of labelling is pressed 1 * 10 ⁷Cell/ml suspends, and before being expelled to the receptor rat, has or do not exist the Mus α of the Chinese People's Anti-Japanese Military and Political College _dCarry out precincubation under the situation of antibody and a kind of IgG1 of contrast antibody.Every used final antibody concentration of animal is adjusted into 5.5mg/kg or about 1mg/ animal.At least do 4 animals under every kind of condition.

The receptor rat is weighed, and the ACE solution (containing 0.25ml ketamine, 0.2ml ACE, 0.8ml Rompin) of injection 150 to 200 μ l is anaesthetized under the percutaneous.To every kind of antibody treatment, to animal via intravenous injection 1.0 * 10 ⁷Cell.The every kind of cell suspension that uses gamma counter to detect about 300 μ l is determined total injection cpm/ rat.Allow the NK cell of labelling in rat, to circulate 3 hours, put to death animal then, extract the peripheral blood of 1.0ml with the aorta puncture method.Take out spleen from every animal, weigh, and count with gamma counter.For mensuration is got back to the cell percentage ratio of spleen, with count per minute (cpm)/spleen divided by the known total cpm that is expelled in the rat.Be to measure the cpm in the peripheral blood, suppose that blood accounts for the about 6.0% of rat gross weight, multiply by 6.0% animal gross weight with the cpm in the 1.0ml blood and determine total cpm in the blood.Then with these data divided by the total cpm that is expelled in every animal, the percentage ratio of the cpm that obtains to keep in the blood.

In first experiment, antibody 226B, 226G, 226H, 226I, 20C5B (a kind of non-barrier CD18 antibody) and a kind of control antibodies have been used.Two kind of 226 antibody of antibody 226B and 226G and control antibodies and other is compared, and can obviously reduce the cell quantity of getting back in the spleen.Get back to spleen with about labeled cell of 7% to 8% behind the control antibodies incubation, and with 226B and 226G incubation after about 6% cell get back to spleen.The percentage ratio of total cpm in the blood is between 0.9% and 1.4%, and except 226B, the total cpm percentage ratio of the blood between the different disposal group does not show evident difference, and the 226B group is lower than the value of other groups.

In second experiment, top definite program several adjustment have been done.At first, 4 animals of every kind of condition are increased, the second, adopting property transferase 12 .5 after week rather than above after 4 weeks of introducing, from tumor-bearing rat, take out spleen.Prepare the NK cell by accurate identical method, and by being expelled in the receptor behind top definite dosage and antibody 226B or 226G or a kind of control antibodies incubation.Equally, the cell cycle of permission labelling 3 hours is as above put to death animal then, collects blood and spleen.In addition, from two animal/environment, take out tissue, measure other positions of tumor cell.These tissues comprise liver, brain, thymus, lung, long bone (for bone marrow) and kidney.

The result shows, in contrast IgG1, about 32% tumor cell is positioned at spleen, and the labeled cell of spleen all shows and the quantity of minimizing is respectively 28% and 29% among 226B and the 226G.The percentage ratio of cell is all similar to every kind of antibody in the blood, has found total cpm of 3 to 4% in blood, and the 226G antibody treatment is slightly less than other two groups.

The tissue distribution of each processed group is similar, and liver shows 27% total cpm, brain 0.05%, thymus 0.10%, lung 15%, kidney 0.80%, long bone 1.3%.

In the 3rd experiment, increase the significant difference that size of animal (n=6 or 7) under every kind of condition manages to detect top three processed group.Equally, use and take from the spleen of experiment the last fortnight, and be prepared with the same procedure of introducing above with the animal of tumor cell injection.Cell carries out labelling in a like fashion, is expelled in the animal and allows to circulate 3 hours.In this experiment, because the size of animal that uses is big, and only collect blood sample and spleen from animal.

The result is consistent with experiment for the second time, and about 30% labeled cell is observed and gets back to spleen in matched group, and has only 25%, has only 27% to be found in the spleen in the cell of 226G antibody treatment at the cell of 226B antibody treatment.Blood values does not show main difference equally between each group, total cmp of about 17% is found in the blood in matched group, and 226B and 226G processed group find 15.8% and 14.75% respectively.

For determine 3 hours whether be the optimum time point that detects the processed group differences, in the 4th experiment, done very little adjustment.Equally, use the same method and separate and the preparation cell, be used for being expelled to receptor, except another anti-CD18 antibody 20C5B is joined in the sequence of antibody to be measured.In addition, each condition is only used 4 animals.In this experiment, cell only allows circulation 30 minutes after injection, extracts blood and takes out spleen from animal on this time point.

On 30 minutes time points, the total cpm in the spleen reduces to 12% to 13% from observed value experiment for the second time and experiment for the third time.Processed group all in the spleen sample do not have evident difference, though two in four animals of antibody 226B processed group have lower slightly value.Blood values between all groups is similar equally, finds total cmp of about 6 to 7% in blood.Unique notable difference between each blood group is the bigger diffusion of numerical value from 226B and 226G antibody treatment animal.These discoveries show, α _dReturning nest the leukaemia works to spleen.Experiment shows that returning nest needs a few hours, and uses α _dThe maximum of monoclonal antibody specific suppresses to occur in 3 hours.

Multiple sclerosis and atherosclerotic model of rhesus monkey

Immunocytochemical stain and immunoprecipitation show, monoclonal antibody 212D and 217L can with the cross reaction of macaque splenocyte.The specificity of this identification is by a kind of α from the macaque spleen _dThe immunoprecipitation and the aminoterminal order-checking of planting congener confirm (embodiment 34).Because these former observations, these two kinds of antibody are used to the tissue available from the macaque of experimental autoimmune encephalomyelitis (EAE) or atherosclerosis study is dyeed.The sign of these two kinds of diseases all be engulfing property macrophages infiltration in focus, EAE be the picked-up myelin basic protein (MBP), be low density lipoprotein, LDL in atherosclerosis.MBP or fat pile up that macrophage can (Dako, Carpinteria CA) dye and differentiate with morphology or with Oil Red (ORO) or antibody Ham 56.Used method is same as to introduce among the embodiment 18 and analyzes α in these researchs _dThe used method of expression characteristic in people's tissue.

Feature from the section of the macaque brain of suffering from EAE is the infiltration of lymphocyte and macrophage.α _dBe arranged in a subgroup of focus macrophage in expression, these macrophages can dye with ORO, have absorbed MBP before showing.The negative focus of ORO dyeing also is α _dExpress negative.This result has shown ORO dyeing and α _dContact directly.Also observe similar result with antibody 217K, 217I with 217H.

The arteriosclerosis focus is available from thoracic aorta or the ventral aorta of the macaque of raising with food rich in fat.Focus occurs in two positions in the people, but the focus of generation pathology more generally is positioned at ventral aorta.The focus of test is divided into 5 different stages (I to V) and normal in this research.Stage (IV/V) focus is from ventral aorta, and all the other are from thoracic aorta.

Early stage focus (I/II) shows seldom macrophages infiltration and low-level or do not have a α _dExpress.In the later stage focus, foam cell soaks into and increases, and detects α _dExpress.

In two tissues, the dyeing collection of illustrative plates and the α of other leukocyte beta 2 integrin alpha chain subunits _dExpress overlapping but inequality.Be non-α the most significantly _dAnti-α is used in being expressed in of the α subunit of leukocyte integrin _dAntibody can not detect on the painted lymphocyte.

These results show, in two kinds of animal models, and α _dExpression may be the feature of engulfing property macrophage.But not clear, α _dWhether participate in directly and engulf or some downstream process antigen presentation for example.

Embodiment 37

α _dExpression in preclinical models

For estimating α _dDifference in the various disease state is expressed, and uses the anti-α for preparing by the method for introducing above (embodiment 22) from the tissue slice of animal disease model _dPolyclonal serum dyeing.It is thick to be cut into 6um from the tissue of normal and disease rat, and on Superfrost Plus (VWRScientific) microscope slide in the air at room temperature dried overnight.After the drying, the section be stored in-70 ℃ standby.Before the use, microscope slide was placed about 5 minutes at 50 ℃ from-70 ℃ of taking-ups.To cut into slices and in cold (4 ℃) acetone (Stephens Scientific), at room temperature fix 10 minutes, and in drying at room temperature.Each section contains among the 1X TBS of 30% normal rat serum (HarlanBioproducts), 5% normal sheep serum (Vector Laboratories) and 1% bovine serum albumin (BSA) (Sigma Chemical Company) with 150 μ l at room temperature sealed 30 minutes.Leniently go up sucking-off solution then from cutting into slices.With rabbit polyclonal serum and the preimmune serum of taking from same rabbit in lock solution respectively dilution be protein concentration 34 μ g/ml and 38.5 μ g/ml, and get 100 μ l respectively and join in each section, placed 30 minutes for 37 ℃.Go up sucking-off serum from cutting into slices, and by in 1X TBS, washing three times to remove unconjugated antibody, each 5 minutes.Last washing back is inhaled and is removed unnecessary TBS.The program of recommending according to manufacturer from the biotinylated goat anti-rabbit antibody of an Elite rabbit igg Vectastain_ABC test kit (Vector) is prepared, and the solution 100 μ l that obtained are used for each section, places 15 minutes for 37 ℃.Microscope slide is washed twice in 1X TBS, washs 5 minutes at every turn, and the streptavidin-Jin conjugate (Goldmark Biologicals) that will dilute at 1: 100 in 5% normal rat serum and 1%BSA is used for each section then, and room temperature was placed 1 hour.Microscope slide washs 5 minutes at every turn, and adds the TBS buffer that 100 μ l contain 1% glutaraldehyde (Sigma), room temperature 5 minutes with TBS washing three times.Microscope slide washs three times in TBS again, washs 5 minutes at every turn, and washs 5 times in sterile deionized water, washs 3 minutes at every turn.Excess liquid is gone from each microscope slide suction, in every section, add two silver and strengthen and starting soln (Goldmark Biologicals).Reaction was at room temperature carried out 20-30 minute, the abundant rinsing of will cut into slices then in sterile deionized water, and air-dry overnight at room temperature, and with Cytoseal 60 (VWR) sealing.In contrast, section is carried out labelling with identical method with the monoclonal antibody of identification CD11a, CD11b, CD11c and CD18 in same experiment.

Use α _dPolyclonal serum and at the labeling of monoclonal antibody of CD11a, CD11b, CD11c and CD18 has shown α _dWith observed different dyeing collection of illustrative plates in other α subunits.

In normal lung tissue, α _dBe expressed in that bronchial respiratory epithelium (but not being the epithelium in alveolar gap) is gone up and seemingly be detected on the individual cells of pulmonary alveolar macrophage in the air gap.With the observed signal of polyclonal serum apparently higher than the background signal level that arrives with the preimmune serum controlled observation.In lung granuloma tissue, giving polysaccharide 24 and after 96 hours, using α _dThe respiratory epithelium of dyeing alveolar region detects different signals, and detects stronger signal on the part of trachea macrophage seemingly.In the lung tissue of the animal that in disease, recovers (putting to death in 16 days after the polysaccharide administration), use α _dAntibody is not observed signal.In these tissues, observe few background signal with preimmune serum.

Use is used α from the lung tissue of rats of the asthmatic model of antigen induction _dAntibody has detected very strong signal in the respiratory epithelium in bronchus and alveolar crack.The background signal level of this signal in the preimmune serum.

Preclinical models---Listeria monocytogenes

Evidence shows, α in the splenic red pulp _dPositive macrophage participates in removing impaired erythrocyte and other granules from circulation.Bacterial components is also by the α in the splenic red pulp probably _dPositive macrophage is removed from circulation.Not needing a kind of T cells with antigenic specificity to reply inductive non-infectious material will directly be removed by the red pulp macrophage.On the contrary, the opportunistic infection material is removed by the red pulp macrophage needs the T cellullar immunologic response that produces for eliminating bacteria really.Therefore the α that expresses on might the red pulp macrophage _dMove to the marginal zone or help regulate macrophage/T cell interaction from red pulp by regulating macrophage by causing the accessory molecule of the macrophage/T cell interaction of t cell activation to work as participation.

Be research α _dTo the effect in the infectious substance immunne response, use a Listeria monocytogenes mouse model to α _dExpression in spleen is estimated.To the α on the red pulp macrophage of having engulfed antibacterial _dExpression detects.At α _dAntibody also in this model, detect, determine α _dRole in inducing at the t cell response of Listeria monocytogenes.

Embodiment 38

α _dEffect in spinal cord injury

After central nervous system (CNS) wound, immunne response relates to mixing [Means, et al., the J.Neurophathol. ﹠amp of invasive neutrophilic granulocyte, natural killer cell and engulfing property monocyte/macrophage; Exp.Neurol., 42:707-719 (1983)].This is replied and comprises that release inflammatory mediator, induced reaction microglia, platelet are soaked into, endothelium destroys and vascular permeability strengthens and edema takes place.Nearest observation prompting, inflammation causes chronic defective after the wound in the spinal cord, and part is destroyed [Blight, A.R., Central Nervous System Trauma, 2:299-315 (1985)] by demyelination or by more direct neuron and aixs cylinder.In addition, nearest research report, the disorganization amount significant correlation of each level of spinal cord behind the number of macrophage/microglia and the impact damage [Carlson, et al., Exp.Neurol., 151:71-81 (1998)].Neutrophilic granulocyte and macrophage phagocytic fragment are induced oxidative burst again, cause producing reactive oxygen.Although these antibacterial agents are effective, cause the destruction of surrounding health tissue.Therefore, the generation of lymphocytic infiltration and the reactive oxygen of following participates in sending out of initial bump site secondary injury in addition.This hypothesis supported by some institutes, and neutrophilic granulocyte or macrophages infiltration cause the degree of injury minimizing after apoplexy or the spinal cord injury in this research.

Complete cross-section model

In order to assess α _dCross-section model of rat and α have been used in effect in spinal cord injury _dMonoclonal antibody, wherein some the blocking-up α _dWith combining of its part VCAM-1.In this model, induced cross-section fully at fourth dorsal vertebra (T) sections, this has produced autonomic reflex obstacle [Krassioukov, et al., Am.J.Physiol.268:H2077-H2083 (1995)].The advantage of this model is the narrow section of former the disorganization that remove on the living border of the little surgery diseases sell of one's property, and it helps analyzing the influence of spinal cord injury to this zone.

All experiments are all formulated " laboratory animal nursing and guide for use " middle principle of establishing according to Canadian Animal Protection Association and are carried out.At first give male Wistar rat (Charles River) atropine (0.5mg/kg) and stable (2.5mg/kg) that body weight is the 270-320 gram by peritoneal injection.After 10 minutes, by peritoneal injection sodium phenobarbital (35mg/kg) anesthetized rat.Replenish injecting narcotic (2mg/kg) during operation as required.During operation rat is placed on the pad of heating, its body temperature is remained near 37 ℃.In the back projection of microscopically removal third dorsal vertebra (T3), carry out laminectomy with exposing spinal cord.With scalpel at the complete cross-section spinal cord of T4 segments of spinal cord.Close muscle and skin on the laminectomy position, animal is recovered under thermolamp.The postoperative care of paraplegia rat was carried out [Krassioukov et al., Neurosci.70:211-226 (1996)] according to former description.After from operation, recovering, arbitrarily provide food and water.Animal survived two days after surgery.

Rat is divided into following treatment group (4-5/group): (1) 1mg/kg α _dMonoclonal antibody 226H, 236L, 226B, or IgG1 κ antibody 1B7 and (2) 5mg/kg α of irrelevant isotype coupling _dMonoclonal antibody 226H, 236L, or contrast 1B7.In therapeutic scheme, every mice is only accepted a kind of antibody.

Use the recombined human VCAM-1 that merges with immunoglobulin domain and express rat α _dSelect antibody according to external in conjunction with measuring with the Chinese hamster ovary celI system of people CD18.Detected each antibody blocking α in one group of antibody _dWith the bonded ability of VCAM-1.Show that in conjunction with the result who measures some antibody are not blocked in conjunction with (" non-blocker "), some block (" medium blocker ") in 50% scope, and other blocks (" strong blocker ") in the scope of 75%-85%.The purposes of from non-blocking-up, medium blocking-up and strong blocking-up group, selecting representative antibodies to be used for hereinafter describing.

Give all monoclonal antibodies in preceding 24 hours of operation, operation back 2 hours and 24 hours by tail vein injection.In the phosphate-buffered saline of pH7.2, dilute antibody, wherein do not contain calcium chloride or magnesium chloride, be convenient to injection to guarantee enough volumes.In another matched group, fully gave the methyl meticortelone that concentration is 30mg/kg (MP) by tail vein injection in cross-section back 30 minutes at spinal cord, spinal cord cross-section back 2 hours and 24 hours administration concentration fully is 15mg/kg.

Postoperative two days, before heart perfusion by peritoneal injection 3g/kg urethane (Aldrich Chemical Company, Inc., Milwaukee, WI is USA) with the animal deep anaesthesia.After opening the thoracic cavity, heparin is injected to left ventricle.Oxygenate tissue culture medium (TCM) (Dulbecco ' s modified Eagle medium with 250ml pH7.4; Gibco BRL) the perfusion rat is dissolved in 4% formaldehyde fixed perfusion in the 0.1M phosphate buffer (pH7.4) with 500ml then.The thoracic vertebra (T4-8) of removing cross-section place tail end as previously mentioned is used for checking [Krenz andWeaver, Neurosci 85:443-458 (1998)].After in identical fixative, fixedly spending the night, with 4 ℃ 10%, 20% and 30% the freezing preservation spinal cord of the sucrose solution part that is dissolved in PBS.On cryostat, spinal cord partly is cut into dropping cut slice (50 μ m) then.To section statining, manifest polymorphonuclear leukocyte with 1% cresol-purple of pH4 with standardization program, rinsing in dimethylbenzene, (BDH Laboratory Supplies, Poole U.K.) cover to soak tablet with DPX.Count with the neutrophilic granulocyte that shows the leafy nuclear of characteristic showing circular macrophage/microglia of engulfing profile after the cresyl violet stains in the spinal cord zone in pathological changes site.With bright-field microscope with have a grid (gross area 0.08mm ²) the 40X object lens to carry out immunocyte quantitative.Check the immunocyte of three sample areas, from cross-section spinal cord edge, caudal ward moves, and from one side to another side.This program causes 2.72mm ²The average total quantitatively area of spinal cord.In another spinal cord slice, repeat this process then.Select sample area (border is between V and VII) from the dropping cut slice of spinal cord, because inflammatory reaction is the most remarkable in grey matter with maximum grey matter.The macrophage of counting in each sample area and the sum of neutrophilic granulocyte are divided by gross sample area (mm ²) obtain every mm in each treatment group ²Macrophage and neutrophilic granulocyte number.Relatively Antybody therapy group (1mg/kg and 5mg/kg) and same dosage indifference IgG separately mate matched group and the cross-section matched group of non-drug therapy.In further relatively, the group of the average number of the most remarkable reduction immunocyte is treated animal with MP and the operating comparison animal compares.Under the situation of not knowing the treatment group, carry out the counting of all cells by blind method.

The result shows that two days later cross-section, activated microglia and/or blood macrophage are accredited as mononuclear cell, and the round cell with transparent maxicell matter in the pathological changes site clearly.Find not accept α _dThe control animal of antibody or corticosteroid treatment is at the every mm in spinal cord injury site ²Average macrophage/microglia number be 396 ± 26.The treatment of the irrelevant IgG1 antibody 1B7 of low dosage (1mg/kg) makes macrophage/microglia number be reduced to 362 ± 43/, but this value does not have significant difference with the control animal of not accepting Antybody therapy.α in check _dIn the antibody, the 226H of 1mg/kg compares with cross-section contrast with 1B7 antibody with 236L and all causes every mm ²The macrophage average number significantly reduce.Particularly, compare with the control animal of not receiving treatment with 1B7 antibody, the 226H administration makes every mm ²Macrophage/microglia number be reduced to 147 ± 17, the 236L administration makes every mm ²Macrophage/microglia number be reduced to 131 ± 4.On the contrary, the α of injection 1mg/kg _dAntibody 226B makes every mm ²Macrophage/microglia number be reduced to 327 ± 65, this compares with the value of matched group does not have significant difference.The MP treatment makes every mm ²Macrophage/microglia number be 250 ± 24, this compares remarkable minimizing with control animal, but than with 226H and 236L α _dDetected value is high in the animal of Antybody therapy.This result do not reckon with, because methyl meticortelone is the most widely used medicine in the clinical treatment of acute spinal cord injury.

Work as α _dWhen the dosage of antibody increases to 5mg/kg, every mm of 226H and 236L treatment group ²Macrophage/microglia digital display work be lower than matched group, but this number with do not have significant difference with the number in the animal of the control antibodies treatment of irrelevant IgG1 isotype coupling.

Result about neutrophilic granulocyte (NLs) shows that great majority detect in whole grey matter with single or aggregated forms.Have only the visible NLs of sub-fraction in the white matter of the every side of grey matter, to detect.And, find that in myeloid tissue's section some neutrophil adhesion are in venule and arteriolar chamber face.In the animal of not receiving treatment, every mm ²Average N Ls number be 295 ± 56, in animal with 1mg/kg 1B7 treatment, every mm ²NLs digital display work increase to 416 ± 63.Compare α with the control animal of not receiving treatment _dTreatment has significantly reduced the NLs number.Particularly, the 226H of 1mg/kg and 226B make every mm respectively ²NLs digital display work increase to 361 ± 80 and 332 ± 43.Compare with the animal of 1B7 treatment, the 236L of 1mg/kg and MP treatment make every mm respectively ²NLs digital display work be reduced to 270 ± 39 and 193 ± 39.Yet these results compare with the matched group of not receiving treatment does not have significant difference.

At 5mg/kg, the 1B7 treatment makes every mm ²Average N Ls number increase to 343 ± 37, but this result compares with the animal of not receiving treatment and does not have significant difference.Compare with the animal of 1B7 treatment, antibody 226H makes average N Ls number be reduced to 236 ± 38, but this counting is compared with the animal of not receiving treatment and do not had significant difference.On the contrary, 236L makes every mm ²The NLs number be reduced to 190 ± 17/mm ², comparing with the animal of accepting 1B7 treatment does not have significant difference.

These results show, the α of low dosage _dMonoclonal antibody reduces the leukocyte count in the spinal cord that damages, and may be by the interaction of destruction with VCAM-1, and this was with results reported was consistent in the past.Other uses anti-VCAM-1, and the report of the antibody of the known counter receptor of VLA-4 shows that the infiltration of VLA-4 positive cell in brain reduces, and prevents the clinical and pathology sign of experimental allergic encephalomyelitis (EAE).Verified similar anti-TNF alpha treatment can suppress sickness rate and the seriousness of EAE, and a mechanism of action is that the VCAM-1 that suppresses on the spinal cord blood vessel expresses, and causes leukocyte to enter CNS and significantly reduces.Results suggest before these, the lip-deep α of blocking leukocyte _dCan cause observed inflammatory reaction to weaken with the interaction of endotheliocyte or the lip-deep VCAM-1 of glial cell.

Because one of the antibody of blocking-up macrophages infiltration 226H does not block α _dAnd the interaction between the VCAM-1, these results suggest, the suppression mode of antibody can comprise blocking-up α _dAnd the interaction between other binding partners except that VCAM-1.A kind of probability is the rat homologue that has ICAM-R.Observation in the past shows, except that VCAM-1, and α _dAlso combine, but affinity is far below VCAM-with ICAM-R.What is interesting is that as if ICAM-R lack on endotheliocyte, mainly on static mononuclear cell, lymphocyte and neutrophilic granulocyte, express that this has got rid of it and participate in leukocyte-endothelium adhesion under home.Show that ICAM-R works at the initial period of leukocytic cell-cells contacting, ICAM-R participates in the adjusting of the leukocytic cell-cell interaction of LFA-1/ICAM-1.Effect inducing cell in having proved ICAM-R leukocyte interacting is in early days assembled.Destruction causes accumulative initial contact to reduce the efficient of immunne response.Also verified, the interaction of the another kind of ligand L FA-1 of ICAM-R has induced LFA-1 to be converted to its state of activation in iuntercellular contact site.α _dCan work in an identical manner with the coexpression of ICAM-R on static leukocyte, the interaction between two kinds of albumen may promote to contact dependency leukocyte activation incident.On the contrary, for example, by using α _dAntibody destroys and interacts, and can explain that leukocyte enters the minimizing of spinal cord.

Some explanations can illustrate the increase of macrophages infiltration.The most simply, the possibility of result is system's phenomenon.Perhaps, more the antibody of high dose may cause the crosslinked of Fc γ R on the full-brown macrophage, the initial outburst that causes chemotactic factor to produce, and this attracts extra leukocyte to injury site.In an identical manner, the generation of chemotactic factor can be explained in most cases the more of the neutrophilic granulocyte at the injury site place, 1B7 treatment back that occurs and macrophage.The possible explanation of another of observed result is the variation between animal, because checked α in the outbreeding strain of Wistar rat _dAntibody.For example, on the same group rat may not have slight different immunocompetence state.In order to check this probability, in identical or relevant nest animal, repeated experiment at one time with the monoclonal antibody of low dosage and high dose.

Pincers compressive damage (CCI) model

Another animal model that is used for spinal cord injury is more relevant clinically pincers compressive damage (CCI) model [Rivlin, et al., Surg.Neurol.10:38-43 (1978); Maiorov, et al., J Neurotrauma 15:365-74 (1998)].In brief, prepare rat about the description of complete spinal cord transection, keep spinal cord by carrying out the dorsal part laminectomy in T3 and T4 breast section by the front.With the aneurysm pincers compression spinal cord of improvement, opening clamp under the spinal cord between the Dorsal root of T3 and T4.Then icarceration is closed 1 minute time, open thereafter and clamp and take off.Close rat by stitching then.These aneurysm pincers have carried out special improvement, make them have the power of calibration.In this way, can improve, moderate serious or slight spinal cord injury to pincers to induce.Serious, the moderate of spinal cord injury or slightly clamp all Pathophysiology aspects that compact model accurately and has as one man duplicated people's spinal cord injury comprises ischemia.

Carry out all experiments with above-mentioned program about animal care and treatment administration.Rat in order to one of following scheme serious CCI of treatment (50g) after after the damage 2,24 and 48 hours: (i) anti-α _dMonoclonal antibody 217L (1mg/ml), (ii) anti-α _dMonoclonal antibody 226H (1mg/ml), (iii) anti-α _dMonoclonal antibody 236L (1mg/ml), (iv) isotype control antibodies 1B7 or (v) PBS (do not have treatment contrast).The 6th group of rat accepted 30mg/ml methyl meticortelone (MP) in 24 hours behind CCI, accepted 15mg/ml MP behind CCI in 24 hours and 48 hours.4-5 rat of each treatment group check.At 72 hours, put to death animal, with the fixative perfusion, remove spinal cord thereafter and further handle.Analyzed anti-α _dMonoclonal antibody (217L, 226H and 236L) reduces ED1 ⁺(being monocyte/macrophage) and MPO ⁺The effectiveness that (being phagocyte) cell soaks into to injury site.Identify ED1 by the two stainings of the immunocytochemistry of using two kinds of different fluorogens ⁺And MPO ⁺Cell.Because be difficult to distinguish the cell that has neutrophilic granulocyte sample profile and have the macrophage-like profile sometimes, both all are counted as MPO ⁺Cell.Generally, most of MPO ⁺Cell is a neutrophilic granulocyte.Side (usually in the tail side) counting cells in damage.For every animal, the section of one 16 μ m in per 10 successive dropping cut slices that the complete segments of spinal cord of striding damage is cut is counted.This program is counted always total 6-10 the section of every animal.

The result of this analysis represents, and compares with the animal of PBS or contrast 1B7 antibody or 236L Antybody therapy, with the wellability ED1 of antibody 217L and 226H treatment back injury site ⁺Cell reduces 40-55%.Discovery accepts in the control animal of PBS or 1B7 antibody 681.3 ± 47.4 and 641.8 ± 77.8 ED1 are arranged respectively ⁺Cellular infiltration is to the site of serious CCI.Use 217L, the rat of 226H or 236L Antybody therapy shows 388.6 ± 52.8,291.9 ± 67.7 and 692.7 ± 98.4 wellability ED1 respectively in injury site ⁺Cell.Rat with the methyl meticortelone treatment shows 409.0 ± 42.6 wellability ED1 in injury site ⁺Cell.These results' that carry out with two-way Anova statistical results show is with the animal remarkable improvement (p＜0.05) different with matched group of 217L or 226H treatment.Wellability ⁺The similarity analysis of cell shows, and do not treat animal or compares with the animal of control antibodies 1B7 treatment, and 217L has reduced the migration of these cells to injury site the most constantly.Find to use 217L, the rat of 226H or 236L Antybody therapy has 156.5 ± 63.3,191.3 ± 39.0 and 299.1 ± 44.0 wellability MPO respectively in injury site ⁺Cell.Control animal with PBS or 1B7 Antybody therapy shows 615.3 ± 45.7 and 260.9 ± 48.92 wellability MPO respectively in injury site ⁺Cell.Yet because the peak time of neutrophil infiltration was generally behind the CCI about 18 hours, the data that obtain after back 72 hours in damage may not reflect anti-α _dMonoclonal antibody reduces the effectiveness of neutrophil infiltration.

Also carried out adopting the similar experiment of the rat of accepting moderate CCI (35g).This result of experiment shows, accepts the wellability ED1 of injury site in the animal of 217L antibody ⁺Cell and MPO ⁺Cell number significantly reduces.The control animal of accepting PBS or 1B7 antibody shows 832.3 ± 69 and 1075 ± 87.94 wellability ED1 ⁺Cell.Use 217L, the rat of 226H or 236L Antybody therapy shows 507.0 ± 51.4,797.4 ± 65.1 and 761.2 ± 88.2 wellability ED1 respectively in injury site ⁺Cell.The wellability MPO that carries out in injury site ⁺Cytoscopy shows, uses 217L, and the rat of 226H or 236L Antybody therapy has 190.5 ± 28.4,253.7 ± 45.2 and 301.4 ± 36.6 MPO respectively in injury site ⁺Cell.Control animal with PBS or 1B7 Antybody therapy shows 611.7 ± 140.5 and 247.6 ± 18.1 MPO respectively in injury site ⁺Cell.

Anti-α _dThe rat motor scoring of mAb treatment

Description by the front is carried out serious CCI and is used Antybody therapy 4 rats.After damage the 1st, 7,10,14,17 and 19 days, by Basso, the assessment of the motor capacity that Beattie, 21 marking systems of Bresnahan (BBB) [Basso, et al., J.Neurotrauma 12:1-21 (1995)] recover rat.In brief, the BBB marking system is based on three Main Stage recovering after the spinal cord injury.Stage is observed and is not had or seldom have hind leg to move in early days, and the interstage is characterised in that inharmonic taking a step, the late stage animal show toe and tail slowly and the motion of effort, the unstable and sole of trunk rotates.Each of three phases is further divided into the classification that is characterised in that specific behavior.Assessment rat 41C450 and 42C50 in 19 days time period, assessment rat 44C50 and 45C50 in 17 days time period.The horizontal bar of assessment in 14 days represents to have the average BBB scoring that rat reaches of not treating of identical major injury.The scoring of each rat both sides represents that compressive damage is uniformly applied to spinal cord.

In 4 rats being checked 3 with do not treat rat and compare the clearly increase that shows exercise recovery.When should be noted that 17 days, all 4 rats have also recovered bladder control to a certain degree.

The autonomic reflex obstacle of assessment CCI rat

Assessed the autonomic reflex obstacle [Maiorov, D.N.et al., J.Neurotrauma 15:365-374 (1998)] of above-mentioned 4 rats by the expansion colon.Rat 44C50 and 45C50 began to carry out this assessment at the 17th day, rat 41C50 and 45C50 were beginning in the 19th day.Record when beginning successive two days, to obtain twice heart rate (HR), arterial pressure (AR) and mean arterial pressure (MAP) arterial cannulation day.The rat 217L antibody that gives major injury makes ectocolon reduce about 50% to the influence of arterial pressure.Untreated injury rats shows MAP has on average increased by 42 ± 3mmHg than the basic blood pressure of about 100mmHg.On the contrary, show MAP with the rat of the identical damage of 217 Antybody therapies and on average increase by 22 ± 3mmHg.The average heart rate (HR) of untreated tranquillization CCI rat be 515 ± 16 jump/minute.After inducing the autonomic reflex obstacle, during ectocolon the HR value reduced 124 ± 13 jump/minute.With the average heart rates of 4 tranquillization rats of 217L Antybody therapy be 544 ± 9 jump/minute.After inducing the autonomic reflex obstacle, ectocolon causes this value to reduce 117.7 ± 18.Therefore before inducing the autonomic reflex obstacle and treatment afterwards and the HR that do not treat rat do not have remarkable significant difference, this may be because to react on the alteration in heart rate of ectocolon be by not relying on the parasympathetic vagus nerve that spinal cord injury works.

After after the damage 12,24 or 48 hours, check and use anti-α _dThe rat of mab treatment

If anti-α _dThe first administration of monoclonal antibody can be delayed to after the spinal cord injury 12,24 or 48 hours and still effective, will be useful, will enlarge because give the time range of effectively treatment.In order to study this probability, according to description (i) PBS, (ii) 1B7 antibody or (iii) anti-α above _dMab treatment has been accepted the rat of serious or moderate CCI, and (then behind CCI 48 hours injection of antibodies) again gives antibody just 12 hours (then in 24 and 48 hours injection of antibodies behind the CCI) behind the CCI or 24 hours.Also carry out similar experiment, wherein just behind CCI, given one time antibody in 48 hours.With method assessment ED1 mentioned above ⁺And MPO ⁺Cell number.If anti-α _dAntibody administration is deferred to behind the CCI 12,24 or 48 hours, and animal shows wellability ED1 ⁺And MPO ⁺The remarkable minimizing of cell number uses this antibody to begin by more late time point after damage so.

Compare the anti-α of single injection _dMonoclonal antibody and three infusion protocol ED1 ⁺And MPO ⁺Cell is to the effectiveness of the infiltration that reduces injury site

The antibody of use single dose will be than favourable with three dosage method.Single-dose treatment will reduce cost, simplification treatment procedure and also can reduce the generation of possible side effect.Based on above-mentioned result of experiment of carrying out, determined the Best Times of antibody administration.Behind CCI 2 hours or giving the anti-α of rat single injection that accepts serious or moderate CCI by analyzing the Best Times that aforementioned result determines _dMonoclonal antibody, contrast 1B7 antibody or PBS.Put to death rat in back 72 hours in damage, measure the ED1 of injury site ⁺And MPO ⁺Cell number.Relatively accept single dose animal the result and with the result of the animal of single dose scheme treatment, to determine the effectiveness of low dose therapy.

Check anti-α _dMab treatment reduces the effectiveness that soaks into to the neutrophilic granulocyte peak of injury site

The normal peak of neutrophil infiltration betides back 18 hours of damage, is reduced to the much lower level of keeping then rapidly.For determining anti-α _dWhether Antybody therapy can reduce the infiltration of neutrophilic granulocyte to injury site, uses anti-α in CCI (serious or moderate) back after 2 or 12 hours _dAntibody, contrast 1B7 antibody or PBS treatment rat.Measure the neutrophilic granulocyte number that soaks into to injury site then, to analyze anti-α in the infiltration of neutrophilic granulocyte peak _dThe effect of antibody administration.Accept CCI and using anti-α _dCheck motor capacity and autonomic reflex obstacle in the rat of Antybody therapy

According to foregoing experiment, determine therapeutic regimen.Make rat accept serious or moderate CCI, adopt therapeutic regimen, with PBS, contrast 1B7 or anti-α _dAntybody therapy.The damage back was used the motor capacity of foregoing standard BBB marking system assessment rat in per 3 days.3 weeks behind the CCI, the autonomic reflex obstacle of a subgroup animal of check, and remaining rat is carried out the motor capacity assessment in other 3 weeks.The assay method of autonomic reflex obstacle as previously mentioned.

Embodiment 39

α _dExpression in clone disease

Work in the past (embodiment 18) shows, has detected higher levels of leukocyte integrin in clone disease patient's tissue slice.In order to assess α in the clone disease _dExpression is subjected to regulating degree, the tissue slice that obtains from ill and normal colon of having checked as mentioned below.

The tissue slice that will obtain from 5 clone disease patients' colon and normal colon be the thickness of 6 μ m, under the room temperature on Superfrost Plus (VWR Scientific) microscope slide dry 5 minutes.Microscope slide is stored in-20 ℃, up to measuring.Between using, about 2 minutes of 50 ℃ of incubation microscope slides.To cut into slices and in cold (4 ℃) acetone (EM Science), fix 2 minutes, at room temperature air-dry.Under the room temperature section placed and contain 100ml 1X TBS, 1.1ml 30%H ₂O ₂(Sigma) and 1.0ml 10%NaN ₃(Sigma) in the buffer 15 minutes, to remove endogenous peroxidase activity.With 20% normal human serum (Boston Biomedica) of each section in containing 1X TBS, incubation is 30 minutes in the 150 μ l sealing buffer of 5% normal rat serum (Harlan) and 2%BSA (Sigma) under the room temperature.Protein concentration with 10 μ g/ml in lock solution prepares first antibody, at room temperature 75 μ l is applied to each section 1 hour.Behind the incubation, each section of washing is 3 times 5 minutes in 1X TBS, to remove unconjugated antibody.Excessive TBS is removed in the washing back around drawing tissue the last time.In the sealing buffer,, at room temperature 75 μ l are applied to each section 30 minutes with 1: 400 dilution biotinylated rat anti-mouse antibody (Jackson Laboratories).Each microscope slide of washing is 2 times in 1X TBS, each 5 minutes.Prepare the link coupled avidin/biotin complex of peroxidase (Vector Laboratories) by the 9 μ l gel reagents A and 9 μ l gel reagents B to the 782 μ l 1X TBS that are provided by manufacturer are provided, at room temperature the mixture that 75 μ l are obtained is applied to each section 30 minutes.Each microscope slide of washing is 2 times in 1X TBS, each 5 minutes.Use substrate 3,3 '-diaminobenzidine (DAB) (Vector Laboratories), stop colour developing in the water by immersing.1 1% osmic acid (VWR) is applied to each cut into slices about 15 seconds,, stop colour developing in the water by immersing with enhancing signal intensity.In Gill ' s hematoxylin #2 (Sigma), negative staining is carried out in section, in water, clean, then dehydration and fixing with Cytoseal (VWR).

In normal colon, use antibody 217L, 217K or 212D detect less than labelling.Antibody 240I label aggregation in lymph node many cells and be dispersed in lymphocyte and eosinophilic granulocyte in the lamina propria.Antibody 240I is seemingly macrophage or activated lymphocytic cell type of labelling also.Similar with the dyeing that antibody 169A carries out to antibody 240I.Antibody 240I labelling be dispersed in lymphocyte and eosinophilic granulocyte in lamina propria and the tela submucosa, and around tremulous pulse be positioned at the smooth muscle cell of a hypotype of muscularis externa.

For the sample of clone disease, adopt the observed marking mode of each antibody overlapping, but the expression pattern difference.Detect less than labelling with antibody 217K or 212D.In the antibody 240I labelling lymph node differentially expressed granuloma and the lymphocyte in the labelling lymph node arranged on multinucleated giant cell.Antibody 240I labelling is dispersed in lymphocyte and the eosinophilic granulocyte in the lamina propria.Antibody 217L also has a differentially expressed granuloma in the labelling lymph node on multinucleated giant cell.Antibody 217L labelling the lamina propria of 240I institute labelling and a little hypotype of lymphoma in the tela submucosa.The dyeing pattern of antibody 169A is similar to 240I, just the less lymphocyte of 169A labelling.The dyeing pattern similarity of the dyeing of antibody 169B and 169A, just 169B also labelling around blood vessel be arranged in a smooth muscle cell hypotype of muscularis externa.

Embodiment 40

From rat spleen α _d ⁺Cell discharges TNF α

In order to identify the spleen α of a uniqueness _d ⁺Cell subsets produces the ability of cytokine when stimulating, carried out following experiment.

Flank is given 1: 1 emulsion of Lewis rat skin lower injection 100 μ l complete Freund's adjuvants (CFA) in PBS in the back, puts to death animal after 7 days.The results spleen prepares single-cell suspension liquid with standardization program.With anti-CD4 (the antibody W3/25 that is installed in advance on the anti-mice IgG magnetic bead conjugate, E.A.A.C.C.No:84112002), CD11b (antibody OX42, E.A.A.C.C.No:87081803) and CD45Ra/b (antibody OX33, monoclonal antibody selective removal B cell Pharmingen), CD4 ⁺T helper cell and macrophage.CD4 antibody is identified the T cell, and CD11b antibody is identified macrophage, mononuclear cell, granulocyte and natural killer cell, CD45Ra/b antibody recognition B cell, T cell subsets, mononuclear cell, granulocyte and macrophage.Remove the cell of antibody sandwich then with Magnet.Collect non-adhesion T cell, carry out the positive with biotinylated rat anti α d monoclonal antibody 205C and 226G and select, use the streptavidin magnesphere incubation then.With the cell of Magnet collection antibody sandwich, and with 5 * 10 ⁵Cell/ml is suspended in growth medium (RPMI 1640 that comprises 2% normal Lewis rat blood serum, penicillin/streptomycin Sodium Pyruvate, L-glutaminate).

To the 3 Mus CD3 of μ g/ml Chinese People's Anti-Japanese Military and Political College monoclonal antibodies (G418, Pharmingen), irrelevant control antibodies or do not have in each hole of 24 orifice plates of antibody sandwich and add the 2ml cell suspending liquid.Incubation plate in 37 ℃ of following 7%CO2 is collected supernatant from each hole after 20 hours, 48 hours and 72 hours.Five equilibrium supernatant and be stored in-70 ℃ immediately after the collection.Before mensuration,, and place Chinese People's Anti-Japanese Military and Political College's Mus TNF α detection assay equipment (Biosource) with 1: 2 dilution supernatant.

The result shows, after stimulating with CD 3-resisting monoclonal antibody, and α after 20 hours _d ⁺Cell discharges about 280pg/ml TNF α, and antibody control and culture medium processed group discharge about 40pg/ml.

Embodiment 41

Adjusting is from α _dThe activated splenocyte of antibody discharges TNF α

In order to assess α _d ⁺Following experiment has been carried out in the effect of engulfing property splenocyte in inflammatory reaction.

Because shown α in the past _d ⁺Splenic macrophage has been engulfed the magnetic-particle that is injected in the rat, collects such cell in the following ways.Give injection 200 μ l bead suspension (amine is link coupled, Perspective Biosystems) in 4 mouse veins.After 24 hours, take out spleen, prepare single-cell suspension liquid by wire screen by making tissue.Use the Magnet isolated cell, washing once places the culture of RPMI/10%FBS culture medium in the PBS that contains magnesium and calcium, grows under following six conditions: (i) non-processor; (ii) use and mice α _dThe Mus α of the hamster Chinese People's Anti-Japanese Military and Political College of cross reaction _dMonoclonal antibody 205C (10 μ g/ml) handles; (iii) use and mice α _dThe Mus α of the hamster Chinese People's Anti-Japanese Military and Political College of cross reaction _dMonoclonal antibody 205E (10 μ g/ml) handles; (iv) use lipopolysaccharide (LPS) to handle; (v) use LPS and monoclonal antibody 205C (10 μ g/ml) to handle; (vi) use LPS and with mice α _dThe monoclonal antibody 205E of cross reaction (10 μ g/ml) handles.

At first used the antibody treatment cell 30 minutes, and collected the sample of 200 μ l conditioned mediums thereafter, this interval scale start time point (t=0).The LPS (10ng/ml) that adds in the hole as indicated above then collects the five equilibrium thing of culture medium 0.5 hour, 1 hour, 2 hours and 4 hours, (ENDOGEN #005452) measures the TNF α that discharges by ELISA to adopt Mus TNF α test kit.After the collection, freezing immediately sample is up to mensuration.Before mensuration, measure with 1: 1 diluting condition culture medium and according to the scheme of manufacturer's suggestion.

The result represents that no matter whether used antibody treatment, the remarkable TNF α that does not show in culture medium with the activated splenocyte of LPS does not discharge.But in culture medium, discharge the TNF α of detection level with the splenocyte that LPS handles, and show significantly lower TNF α release with the activated cell of LPS of 205C or 205E antibody treatment.These results are consistent at the time point of all checks, and are being confirmed in the multiple mensuration subsequently.In addition, observing identical result with in the experiment of not using the isolating splenocyte of magnetic bead to carry out afterwards.At last, PRELIMINARY RESULTS shows that the IL-1 β of splenocyte discharges by anti-α _dMonoclonal antibody similarly suppresses.

Embodiment 42

Identify the α on the eosinophilic granulocyte _dExpress

Observation in the past shows α _dOn all peripheral blood eosinophilic granulocytes, express (embodiment 18).In order further to check expression and the function on the people eosinophilic granulocyte, carried out following analysis.

α _dThe expression of integrin on human granular leukocyte

On by hereinafter prepared cell, check α _dExpression on human granular leukocyte.Separate eosinophilic granulocyte's (promptly having normal proportion) [Hansel of normal density from volunteer's peripheral blood hypersensitive by the negative system of selection of previously described density gradient centrifugation, hypotonic erythrocyte splitting and immune magnetic greater than 1.09, et al., J.Immunol.Meth.145:105 (1991)].By density gradient centrifugation and independent hypotonic erythrocyte splitting peripheral blood purification neutrophilic granulocyte [Bochner, et al., J.Immunol .145:1832 (1990)] from normal volunteer.The representative purity of cell type surpasses 95% usually.Adopt two percoll density gradient separation enrichment basophilic granulocytes from peripheral blood, this makes the number of basophilic granulocyte increase to the 3-10%[Bochner of total leukocyte counting, et al, J.Immunol.Meth.125:265 (1989)].Adopted according to former description that the assessment of monochromatic indirect immunofluorescence and flow cytometry newly separates, integrin expression [Bochner, et al., J.Immunol.Meth.125:265 (1989) on the cell behind the culture moderate stimulation from blood; Matsumoto, et aL, Blood 86:1437 (1995)].Also carried out the double-colored detection (adopting anti-IgE) of basophilic granulocyte.All samples is fixed in 0.1% paraformaldehyde (Sigma) and is adopted EPICS pattern I I flow cytometer (Coulter) to analyze.Collect about 10,000 incidents, and on the 4-log scale, show, produce the value of average fluorescent strength (MFI).

The result shows that the eosinophilic granulocyte expresses all four kinds of β ₂Integrin.α _dThe surface expression level of integrin is higher than CD11c, but is lower than α _dIntegrin (CD49d), the expression of CD11a or CD11b.The result represents that also basophilic granulocyte is than the α on the neutrality granulocyte _dThe integrin expression level is slightly high.

α on the mediator eosinophilic granulocyte _dIntegrin expression

Carried out the preliminary research intracellular α that determining whether the eosinophilic granulocyte can resemble that neutrophilic granulocyte reported, prompts mobilization _dβ ₂Store [Van der Vierern, et aL, Immunity 3:683 (1995)].With the peripheral blood eosinophilic granulocyte of PMA or calcium ion carrier A 23187 incubation purification (by former description preparation) 15 minutes, measure β by indirect immunofluorescence ₂The surface expression (as previously mentioned) of some α chains of integrin family.

The result shows that the Calcium ionophore of the PMA of 50ng/ml or 1 μ M significantly increases α _dExpression with CD11b.A few minutes in adding PMA, expressing increases, and reaches the level of remarkable increase in 10 minutes.This observed result surface, eosinophilic granulocyte's Cytoplasm α _dβ ₂Storage can prompt mobilization to cell surface, stores similar to CD11b.

According to these results, checked other stimulation that activates the eosinophilic granulocyte to α _dβ ₂The definite effect of expressing.With MDC (100nM), IL-5 (10ng/ml), RANTES (100ng/ml), and eotaxin (100 μ M) incubation eosinophilic granulocyte can not change α in 15 minutes _dIntegrin expression.

Observed result in the past shows, by being exposed to some cytokine for a long time, as IL-5, can strengthen many eosinophilic granulocyte's reactions [Walsh, etal., Immunol.71:258 (1990)] in the phenomenon that is called " exciting ".Therefore designed experiment, be used for check and excite eosinophilic granulocyte's culture whether can cause α with IL-5 _dSurface expression change.The purification eosinophilic granulocyte who prepares as stated above with the 10ng/mlIL-5 incubation 4 days measures the expression of various integrins by the description of front.

Although surface as a result is α _dIntegrin increases 4-5 doubly, α in the expression of cell surface _dThe expression of integrin remains unchanged.α _dThe kinetics that integrin expression increases shows that the expression after cultivating 4-7 days has statistics to be increased significantly.On the contrary, α ₄The level of integrin does not significantly change.PMA exposes back α _dIt is similar to CD11b to express enhanced kinetics, shows that these two kinds of leukocyte integrins may be present in similar or identical cell inner cavity chamber.The position of the chamber of any integrin in the eosinophilic granulocyte is also unknown; Yet in neutrophilic granulocyte, the preformed storage thing of CD11b is positioned specific granule [Todd, et al., J.Clin.Invest.74:1280 (1984); Bainton, et al., J.Exp.Med.166:1641 (1641)].

Because the eosinophilic granulocyte's that bronchoalveolar lavage in late period (BAL) the eosinophilic granulocyte express cell factor excites many features [Kroegel, et al., Ann.Rev.Respir.Dis.143:A45 (1991); Sedgewick, et al., J.Immunol.149:3710 (1992)], also checked α _dExpression on this cell type.From using artemisiifolia or D.petrynissinus extract to carry out obtaining BAL cell [Kroegel, et al., J:Clin.Allergy Immunol.93725 (1994)] the autopath that 18 hours bronchus inner segments anaphylactogens stimulate.Eosinophilic granulocyte's purity in the late period BAL liquid is 19 ± 4%.

The result shows that late period, the BAL eosinophilic granulocyte also showed α _dThe statistics of integrin expression significantly increases, and its level is to similar with the level of IL-5 stimulation culture after 3 days.Check BAL eosinophilic granulocyte in late period, promptly cell adhesion and migration have been carried out so that the α on the cell in the arrival air flue chamber _dDuring the integrin level, observed the expression between the level on new isolating and the eosinophilic granulocyte that IL-5 cultivates.These data show that IL-5 cultivates back α _dAt least a portion of raising of level be because α _dThe increase that integrin is transcribed and translated.

Express alpha _dThe eosinophilic granulocyte combine with VCAM-1

Although proved α _dIntegrin combines with VCAM-1 and possibility mediated leucocytes-leukocyte adhesion [Van der Vieren, et al., Immunit 3:683 (1995)], the α that contrived experiment is expressed on the eosinophilic granulocyte with check _dOther possible part.Part is because former research prompting eosinophilic granulocyte and the β of VCAM-1 ₂Integrin dependency, CD11b dependent/non-dependent adhere to [Matsumoto, et al., Blood 86:1437 (1995)], have carried out preliminary study with immobilized reorganization VCAM-1.

For new purification and eosinophilic granulocyte that cultivate, carried out under 37 ℃ 30 minutes according to former being described in ⁵¹The adhesion [Matsumoto, et al., Blood 86:1437 (1995)] in the hole of Cr labeled cell and VCAM-1 (250ng/ml) or BSA (1%) bag quilt.In some experiments, between check adheres to, with the following closure monoclonal antibody of saturated concentration 4 ℃ of following precincubation cells 30 minutes: anti-CD18 (7E4), anti-CD11a (MHM24), anti-CD11b (clone 44), anti-CD11c (BU-15), anti-α _d(240I) and anti-α ₄(HP2/1).

For transfection with the parental generation Chinese hamster ovary celI, use with plate that the eosinophilic granulocyte adheres to employed identical bag quilt to adhere to.The inspection that transforms Chinese hamster ovary celI is shown α _dExpress lowly relatively, therefore, the interaction between CHO transfectant and the VCAM-1 does not have the interaction between detected eosinophilic granulocyte and the VCAM-1 strong.Therefore used the improvement [Shanley, et al., J.Immunol.160:1014 (1998)] of the soft washing technology of former description.This technology allows that the plate with the centrifugal 30 minutes counter-rotating of 1xg discharges non-adherent cell under 20 ℃.Use 0.1M EDTA (Sigma) to take out remaining adherent cell then, and, except that VCAM-1, in some adhesion experiments, also use E-to select albumen (100ng/ml) bag by the hole by Counting by flow cytometry.The monoclonal anti that is used for eosinophilic granulocyte's research use except sealing is external, handles immobilized VCAM-1 with the suitable diluent of F (ab ') 2 anti-VCAM-1 monoclonal antibodies before adding Chinese hamster ovary celI.

The result shows that new isolating eosinophilic granulocyte adheres to VCAM-1, α _dThe monoclonal antibody blocking-up of integrin effectively suppresses to adhere to.With the not effect of anti-CD11b antibody.Can pass through anti-α _dMonoclonal antibody 240I significantly suppresses to adhere to continuing, although than the anti-α of use ₄The viewed degree of antibody low (approximately suppressing 30%).

Even what more arouse attention is that VCAM-1 adheres to result of experiment, wherein used, and the eosinophilic granulocyte that IL-5 cultivates, it expresses the α of elevated levels _dIntegrin.Under these conditions, the monoclonal antibody of CD18, α _dOr α ₄Integrin equates effectively adhesion to be reduced to background level, and CD11a, the combination of the blocking antibody of CD11b and CD11c is effect not.Also observe, compare with the observed result of new isolating eosinophilic granulocyte, the eosinophilic granulocyte that IL-5 cultivates shows adherent increase of background and the adherent minimizing of VCAM-1.According to the monoclonal antibody blocking-up research of carrying out with new isolating eosinophilic granulocyte, mainly pass through α with the adhesion of VCAM-1 ₄6 integrin-mediated.Yet, in the eosinophilic granulocyte that IL-5 cultivates, α ₄And α _dIntegrin equally mediates the adhesion with VCAM-1.Integrate, these data have at first proved α _dβ ₂The expression of integrin on the people eosinophilic granulocyte and the activation dependency adjusting of function, and write down α _dβ ₂New function as the another kind of part of VCAM-1.

Based on the α on the eosinophilic granulocyte _dThe result that integrin combines with VCAM-1 and can raise with IL-5, this leukocyte integrin can work in the raising of inflammation site the eosinophilic granulocyte of cytokine-stimulated.

Embodiment 43

Express alpha _dChinese hamster ovary celI in conjunction with VCAM-1

In order further to prove α _dβ ₂As the part of VCAM-1, prepared expressing human α as follows _dAnd β ₂The CHO transfectant of integrin chain.

Press the description transfection hamster ovary cell of embodiment 11.Containing 1mM acetone acid and 2mM L-glutaminate (Biofluids), adding in the DMEM/F12 culture medium of 10% FBS, 100U/ml penicillin, 100 μ g/ml streptomycins and 600 μ g/ml G418 (all coming from Life Technologies) and cultivate α _dβ ₂The Chinese hamster ovary celI of transfection.The culture medium that is used to cultivate parental generation Chinese hamster ovary celI system is similar, only is to use the not FBS (Life Technologies) of dialysis, and 0.1mM hypoxanthine and 16nM thymidine (Sigma) have replaced G418.The α of transfected cellular expression moderate level _dAnd β ₂The integrin chain, and do not express CD11a, CD11b, CD11c or α ₄Integrin.Any one in these integrins can not be expressed by parental generation Chinese hamster ovary celI system, adheres to mensuration by the method shown in the embodiment 42.

The result shows, α _dβ ₂The Chinese hamster ovary celI of transfection adheres to the hole of VCAM-1 bag quilt.F (ab ') by anti-VCAM-1 first domain ₂Monoclonal antibody and anti-CD18 or α _dMonoclonal antibody effectively blocked adhesion.On the contrary, the parental generation Chinese hamster ovary celI of untransfected can not adhere to VCAM-1, and two kinds of cell types all do not show with E-selects proteic remarkable adhesion.

About the α in VCAN-1 first domain ₄The monoclonal antibody of integrin binding site is blocked α fully _dβ ₂α is pointed out in the adherent discovery of integrin dependency VCAM-1 strongly _dβ ₂Binding site is adjacent to or is same as α ₄Integrin.Because at α _dAnd α ₄Have only amino acid identity seldom between the integrin, this result is unforeseeable.α _dβ ₂Whether integrin can wait other α with fibronectin or mucosa addressin cell adhesion molecule 1 ₄Integrin ligands is in conjunction with also unknown.

α _dIn conjunction with needed VCAM-1 zone

Have been found that preceding two domain supports and the α of VCAM-1 ₄The related amino acid in these domains has been identified in the combination of integrin.In order to determine α _dHave the recognition site in the similar VCAM-1 molecule, made up the plasmid of the sequence that contains the VCAM-1 domain 1 that is blended in human normal immunoglobulin Fc and 2.In addition, produced the modified forms of two domain expression construct by PCR, replaced sudden change to introduce, wherein the alanine of residue 40 is replaced by asparagicacid residue.With the DEAE-dextran scheme of former description with two expression construct transient transfections to the COS cell.Press former description from the culture supernatants purifying protein with protein A Sepharose_.

Checked express alpha as follows ₄β ₁Or α _dβ ₂Chinese hamster ovary celI and the VCAM-1/Ig fusion rotein or the bonded ability of ICAM-1/Ig fusion rotein of 5 domains.In bicarbonate buffer (pH 9.5), CAMs is fixed in 96 hole microtitration plates with 0.5 μ g/ hole.With 1% isinglass closure plate, with buffer, irrelevant antibody or closure VCAM-1 antibody treatment.With the irrelevant antibody of the monoclonal antibody specific of buffer, α chain (for α _dFor 130K or 217I, for α ₄Be α 4.1) or closure α chain antibody processing α _dOr α ₄The Chinese hamster ovary celI that transforms.Washed cell, the density with 100,000 cells/well adds the hole that CAM wraps quilt then.The incubation cell is 20 minutes in the presence of immobilized antibody, adds 5% glutaraldehyde then.After fixing, use the distilled water wash plate, use 1% violet staining cell then, take off dyeing after several hours, measuring absorbance under the 570nm on the Dynatech plate reader with 66% ethanol.

The result shows, α _dβ ₂And α ₄β ₁With 5 domains and the 2 domain forms of same degree identification VCAM-1, description taken in conjunction may not need other domain.Although at α _d/ VCAM-1 combination and α ₄Detected difference between the/VCAM-12 combination, this difference comes from the differential expression that is transformed in the Chinese hamster ovary celI probably.VCAM-1 antibody (50-100%), α ₄Antibody (100%) and α _dThe combination of two kinds of cell lines of antibody (50%) blocking-up.α ₄Cells transfected is the VCAM1 of nonrecognition sudden change, α _dCell line is with the detected result's of wild type VCAM-1 50% with combining of mutant.Two kinds of Chinese hamster ovary celI systems are not in conjunction with ICAM-1/Ig.Integrate, these results show, α _dAnd α ₄The domain 1 and 2 of identification VCAM-1, and discern overlapping but incoordinate epi-position.

Embodiment 44

With α _dTarget is decided to be tumor antigen

α _dRoom and time limiting expression prompting, this molecule may be as removing surface expression α _dThe target of population of pathogenic cells.Some observed results have in the past caused this probability.

For example, compare α with other leukocyte integrin _dThe expression scope is less.α _dExpression as if be limited to specific and/or well differentiated lymphocyte subtype.Different with other leukocyte integrin, α _dAs if regulated, this was proved by the former generation in cultivating or the rapid downward modulation of transformant.α _dMonoclonal antibody show variable reactivity, although antibody is to be specific to α _d.For example, with another kind of anti-α _dThe observed reactivity of monoclonal antibody 217L is compared, anti-α _dAntibody 212D shows and normal structure and well differentiated myelocytic limited response.What is interesting is, knock out α _dMice survival longer to birth or survival, but this observed result does not almost provide about α _dThe information of biological function.At last, on the dog leukaemia of estimation 70%, detected α _dExpress, detecting high-caliber α from the new isolating NK leukaemia of F344 rat _dExpress (embodiment 26).Although tumor cell can be to use α _dBe the preferred cell group that target is removed, any express alpha _dRequired cell type also can remove in mode hereinafter described.

The preferential antibody of selecting one or more and normal structure to have low level reaction.According to the discussion of front, a probability is antibody 212D.Also selected suitable control antibodies, comprised irrelevant antibody as negative control.Obtain blood and tumor sample from leukemia and lymphoma patient, and, use selected antibody analysis cell surface expression by FAScan, immunoprecipitation and western blot analysis by the immunocytochemistry screening.Then, the detection of positive staining can cause developing other program of sweep-out method.

In a method, cloned the hypervariable region of the selected positive staining antibody in front, and in the scope of people's isotype of complement fixation, expressed.After sub-clone and isotype conversion,, for example pass through the histology and the immunoprecipitation of facs analysis, normal structure by specificity and the reactivity assessed once more mentioned above.In order in human IgG1's scope, to express selected hypervariable region, developed the box gene carrier.In addition, a series of primers have been designed and synthesized, to promote from hybridoma cell line increase hypervariable region [Gavilondo-Cowley, et aL, inHYBRIDOMA, Vol 9 No of antibody interested; 5,1990, Mary Anne Leibert Inc., Publishers, Media, PA, pp.407-417].The antibody that external then check obtains is to determine whether its combination in the presence of complement causes cell death.Preferably, use tumor cell to carry out external test.Blank determination will determine that monoclonal antibody is in express alpha not _dCell culture in whether show identical activity.

Also checked monoclonal antibody, determining in conjunction with whether causing internalization, this illustrated this antibody can with the cytotoxic drug coupling.

Develop preclinical models, wherein checked the cells in vivo cytotoxic activity.In one embodiment, in the F344 rat model, checked identity property (embodiment 26).Alternate model is the SCID/Hu system, has wherein transplanted people's cell.For example, bone marrow U937 cell, Jurkat T cell or human colon carcinoma HTC166 cell are transplanted in the mice, the results tumor is used anti-α _dAntibody (as 212D and 217L) has carried out surface antigen dyeing to tumor.α _dThe detection of expressing causes using in the body antibody to remove tumor.

Embodiment 44

The anti-α of people _dMonoclonal antibody

Be illustrated in antibody repertoire on the filobactivirus and surveyor's monoclonal antibody [Waterhouse, et aL, Nucl.Acids Res.21:2265-2266 (1993) by former description by screening; Parsons, et al., Protein Engineering 9:1043-1049 (1996)].In brief, make up the segmental repertoire of strand Fv (scFv) that is illustrated in phage surface with the functional V genetic fragment that comes from non-immune people's donor.To phasmid, this carrier allows do not having to produce under the condition of sub-clone phage display and soluble scFv with fragment cloning.Mix histidine mark to allow by the rapid purification scFv of nickel chelating chromatography.The use of this library form generally allows separation of human monoclonal antibody in the time that was less than for two weeks.Separated [Marks, et al., J.Mol-Biol.222:581-597 (1991), Vaughan, et aL, Nature Biotechnol.14:309-314 (1996)] by former description.The preferred antibody of identifying specific recognition I domain.

Embodiment 45

Anti-α in the Motheaten mice _dAntybody therapy

[Koo, et al., J.Immunol.147:1194-1200 (1992) in the motheaten mutant mice; Koo.et al., J.Immunol.151:6733-6741 (1993)], autosomal recessive me ^VGene is as the spontaneous existence of point mutation of the hematopoietic cell protein tyrosine phosphatase in the C57BL/6 mice.Homozygote produces chronic myelomonocyte inflammation, and this inflammation comprises the gathering of myelomonocyte in lung and skin, causes interstitial pneumonia, atrophy of thymus gland and T cell and NK cell dysfunction.The medullary cell of these mices can shift inflammatory conditions, shows me ^VSudden change is because the stem cell defect in the myelomonocyte approach.Because α _dBe present in the medullary cell, carried out a kind of program to assess anti-α _dMab treatment is in the inhibitory action that from the normal mouse of motheaten mutant mice bone marrow immunopathogenesis is changed.

From Jackson Laboratory, Bar Harbor, ME obtains C57BL/6J (B6)-me ^V/ me ^VWith they normal+/-compatriot (B6)-+/-mice.Mice is maintained in the bioclean environment, food and water arbitrarily are provided.All mices all are 6-10 ages in week.

The 1st day, all mices accepted to be dissolved in 2 μ g/ml α-NK1.1 antibody PK136 (Pharmingen) of 0.5ml PBS by peritoneal injection.The 0th day, all B6+/-mice uses the 750Rad radiation.Press former description from (B6) me ^V/ me ^VMice results bone marrow.Take out cell from tibia and femur, transfer to additional RPMI culture medium, and at intravenous injection incubation 2 hours before accept radiating mice.Immediately by the peritoneal injection anti-α of 5mg/kg that is dissolved in 200 μ l PBS _dAntibody 205C or irrelevant Antybody therapy mice.Described in the past and mice α _dThe Mus 205C of the purification hamster Chinese People's Anti-Japanese Military and Political College antibody of monoclonal antibody cross reaction.Negative control the most, one group of mice is accepted the saline injection of equivalent.At 4-25 days, every other day treat mice with antibody or saline injection, treat altogether 10 times, detect the change of the weight of animals and disease sign.In a word, every group observation continues two months altogether.

Dying state is the terminal point of measuring.Put to death seriously ill animal.Survival rate in the evaluation group, and with the tissue histologic analysis as α _dThe extra index of Antybody therapy efficient.Carried out observation α _dThe similar experiment of the treatment characteristic of antibody is to replenish Prevention Research described above.

The 35th day, use α _dThe mice of Antybody therapy does not all have death, and 2 death in 9 mices of brine treatment group, remaining 7 merely hit 2 syndromic typical situation has taken place.In the group with irrelevant Antybody therapy, 83 death of merely hitting.

Embodiment 46

α _dExpression on the human leukemia cell

According to cell line, leukemia can be divided into two classes, and myelocyte and Lymphocytic leukemia all can further be divided into acute and chronic for these two types.Because α _dExpression obviously is limited to the myelocytic series cell, supposes myelocyte rather than Lymphocytic leukemia express alpha _dIf first hypothesis is correct, will determine that whether second kind of cell line of research is according to the pathogenic α that causes _dThe difference of expressing, thereby prompting α _dDisease association function on these cells.

Description by embodiment 18 has detected α with antibody 212D and 217L _dExpression on peripheral blood cells.When checking the leukaemia, at first pass through the α of flow cytometry normal marrow cell _dExpress, to set up the baseline of this cell type.According to standardization program with antibody 212D and 217L dyeing patient sample, two kinds of antibody all show with bone marrow in monocytic weak reactivity.Antibody 212D only is the critical positive.

Flow cytometry from the leukaemia of patient's peripheral blood or bone marrow shows, has 212D and 217L epi-position on three kinds of acute myelocytic leukemias (AML) patient's myeloblast and monoblast.On the cell that comes from chronic lymphocytic leukemia (CLL) patient, also observed expression.Also assessed the cell that comes from another AML patient in the more late time, discovery is α _dMale.α _dExpression ratio contrast monoclonal anti height 50-100% on cell, but significantly be lower than CD11a, the expression of CD11b and CD11c.

According to these results, also assessed the U937 cell line that is equal to M-4 phase (with the differentiation standard of M1-M5) AML cell, promptly a kind of myelocytic series leukemia.CD11a, the expression pattern of CD11b and CD11c is similar to AML patient's cell.What is interesting is cell surface α _dProteic existence depends on condition of culture.Rich medium (Iscove ' s improvement Dulbecco ' s culture medium, 20%FBS) support α with high serum levels _dExpress, and minimal medium (RPMI 10%FBS) does not then support.

About on CLL patient's lymphoblast, detecting α _dThe discovery of expressing shows α _dCan in the cell of lymphocyte series, express, with other data consistent (be rat CD5 ⁺Cell and dog CD8 ⁺α on the cell _dExpress).The relative high level expression of other leukocyte integrin on these cells will be got rid of can repeat these cellular assays of trans usefulness α _dFunction, and show that the functional redundancy of this family can compensate the inhibition of a member in these cell types.In fact, the α of these cells _dExpression can be transcribed simultaneously with mistake and be taken place.

Although initial hypothesis, α are not supported in these experiments fully _dThe existence of albumen on myelocyte and lymphocyte series leukemia shows that a lot of patients can be the anti-α that tumor is removed rather than function suppresses by application target _dTreatment.

Those skilled in the art can make various modifications and variations to the illustrative embodiment of listing above.Therefore the present invention only is defined by the following claims.

Sequence table

＜110〉W.M. adds Latin

M. Fan Deweilun

＜120〉Novel Human β 2 beta 2 integrin alpha subunits

<130>27866/36646

<140>

<141>

<150>08/173，497

<151>1993-12-23

<150>08/286，889

<151>1994-08-05

<150>08/362，652

<151>1994-12-21

<150>08/943，363

<151>1997-10-03

<150>09/193，043

<151>1998-11-06

<150>09/350，259

<151>1999-07-08

<160>114

<170>PatentIn?Ver.2.0

<210>1

<211>3726

<212>DNA

＜213〉homo sapiens

<220>

<221>CDS

<222>(3)..(3485)

<400>1

tg?acc?ttc?ggc?act?gtg?ctt?ctt?ctg?agt?gtc?ctg?gct?tct?tat?cat 47

Thr?Phe?Gly?Thr?Val?Leu?Leu?Leu?Ser?Val?Leu?Ala?Ser?Tyr?His

1 5 10 15

gga?ttc?aac?ctg?gat?gtg?gag?gag?cct?acg?atc?ttc?cag?gag?gat?gca 95

Gly?Phe?Asn?Leu?Asp?Val?Glu?Glu?Pro?Thr?Ile?Phe?Gln?Glu?Asp?Ala

20 25 30

ggc?ggc?ttt?ggg?cag?agc?gtg?gtg?cag?ttc?ggt?gga?tct?cga?ctc?gtg 143

Gly?Gly?Phe?Gly?Gln?Ser?Val?Val?Gln?Phe?Gly?Gly?Ser?Arg?Leu?Val

35 40 45

gtg?gga?gca?ccc?ctg?gag?gtg?gtg?gcg?gcc?aac?cag?acg?gga?cgg?ctg 191

Val?Gly?Ala?Pro?Leu?Glu?Val?Val?Ala?Ala?Asn?Gln?Thr?Gly?Arg?Leu

50 55 60

tat?gac?tgc?gca?gct?gcc?acc?ggc?atg?tgc?cag?ccc?atc?ccg?ctg?cac 239

Tyr?Asp?Cys?Ala?Ala?Ala?Thr?Gly?Met?Cys?Gln?Pro?Ile?Pro?Leu?His

65 70 75

atc?cgc?cct?gag?gcc?gtg?aac?atg?tcc?ttg?ggc?ctg?acc?ctg?gca?gcc 287

Ile?Arg?Pro?Glu?Ala?Val?Asn?Met?Ser?Leu?Gly?Leu?Thr?Leu?Ala?Ala

80 85 90 95

tcc?acc?aac?ggc?tcc?cgg?ctc?ctg?gcc?tgt?ggc?ccg?acc?ctg?cac?aga 335

Ser?Thr?Asn?Gly?Ser?Arg?Leu?Leu?Ala?Cys?Gly?Pro?Thr?Leu?His?Arg

100 105 110

gtc?tgt?ggg?gag?aac?tca?tac?tca?aag?ggt?tcc?tgc?ctc?ctg?ctg?ggc 383

Val?Cys?Gly?Glu?Asn?Ser?Tyr?Ser?Lys?Gly?Ser?Cys?Leu?Leu?Leu?Gly

115 120 125

tcg?cgc?tgg?gag?atc?atc?cag?aca?gtc?ccc?gac?gcc?acg?cca?gag?tgt 431

Ser?Arg?Trp?Glu?Ile?Ile?Gln?Thr?Val?Pro?Asp?Ala?Thr?Pro?Glu?Cys

130 135 140

cca?cat?caa?gag?atg?gac?atc?gtc?ttc?ctg?att?gac?ggc?tct?gga?agc 479

Pro?His?Gln?Glu?Met?Asp?Ile?Val?Phe?Leu?Ile?Asp?Gly?Ser?Gly?Ser

145 150 155

att?gac?caa?aat?gac?ttt?aac?cag?atg?aag?ggc?ttt?gtc?caa?gct?gtc 527

Ile?Asp?Gln?Asn?Asp?Phe?Asn?Gln?Met?Lys?Gly?Phe?Val?Gln?Ala?Val

160 165 170 175

atg?ggc?cag?ttt?gag?ggc?act?gac?acc?ctg?ttt?gca?ctg?atg?cag?tac 575

Met?Gly?Gln?Phe?Glu?Gly?Thr?Asp?Thr?Leu?Phe?Ala?Leu?Met?Gln?Tyr

180 185 190

tca?aac?ctc?ctg?aag?atc?cac?ttc?acc?ttc?acc?caa?ttc?cgg?acc?agc 623

Ser?Asn?Leu?Leu?Lys?Ile?His?Phe?Thr?Phe?Thr?Gln?Phe?Arg?Thr?Ser

195 200 205

ccg?agc?cag?cag?agc?ctg?gtg?gat?ccc?atc?gtc?caa?ctg?aaa?ggc?ctg 671

Pro?Ser?Gln?Gln?Ser?Leu?Val?Asp?Pro?Ile?Val?Gln?Leu?Lys?Gly?Leu

210 215 220

acg?ttc?acg?gcc?acg?ggc?atc?ctg?aca?gtg?gtg?aca?cag?cta?ttt?cat 719

Thr?Phe?Thr?Ala?Thr?Gly?Ile?Leu?Thr?Val?Val?Thr?Gln?Leu?Phe?His

225 230 235

cat?aag?aat?ggg?gcc?cga?aaa?agt?gcc?aag?aag?atc?ctc?att?gtc?atc 767

His?Lys?Asn?Gly?Ala?Arg?Lys?Ser?Ala?Lys?Lys?Ile?Leu?Ile?Val?Ile

240 245 250 255

aca?gat?ggg?cag?aag?tac?aaa?gac?ccc?ctg?gaa?tac?agt?gat?gtc?atc 815

Thr?Asp?Gly?Gln?Lys?Tyr?Lys?Asp?Pro?Leu?Glu?Tyr?Ser?Asp?Val?Ile

260 265 270

ccc?cag?gca?gag?aag?gct?ggc?atc?atc?cgc?tac?gct?atc?ggg?gtg?gga 863

Pro?Gln?Ala?Glu?Lys?Ala?Gly?Ile?Ile?Arg?Tyr?Ala?Ile?Gly?Val?Gly

275 280 285

cac?gct?ttc?cag?gga?ccc?act?gcc?agg?cag?gag?ctg?aat?acc?atc?agc 911

His?Ala?Phe?Gln?Gly?Pro?Thr?Ala?Arg?Gln?Glu?Leu?Asn?Thr?Ile?Ser

290 295 300

tca?gcg?cct?ccg?cag?gac?cac?gtg?ttc?aag?gtg?gac?aac?ttt?gca?gcc 959

Ser?Ala?Pro?Pro?Gln?Asp?His?Val?Phe?Lys?Val?Asp?Asn?Phe?Ala?Ala

305 310 315

ctt?ggc?agc?atc?cag?aag?cag?ctg?cag?gag?aag?atc?tat?gca?gtt?gag 1007

Leu?Gly?Ser?Ile?Gln?Lys?Gln?Leu?Gln?Glu?Lys?Ile?Tyr?Ala?Val?Glu

320 325 330 335

gga?acc?cag?tcc?agg?gca?agc?agc?tcc?ttc?cag?cac?gag?atg?tcc?caa 1055

Gly?Thr?Gln?Ser?Arg?Ala?Ser?Ser?Ser?Phe?Gln?His?Glu?Met?Ser?Gln

340 345 350

gaa?ggc?ttc?agc?aca?gcc?ctc?aca?atg?gat?ggc?ctc?ttc?ctg?ggg?gct 1103

Glu?Gly?Phe?Ser?Thr?Ala?Leu?Thr?Met?Asp?Gly?Leu?Phe?Leu?Gly?Ala

355 360 365

gtg?ggg?agc?ttt?agc?tgg?tct?gga?ggt?gcc?ttc?ctg?tat?ccc?cca?aat 1151

Val?Gly?Ser?Phe?Ser?Trp?Ser?Gly?Gly?Ala?Phe?Leu?Tyr?Pro?Pro?Asn

370 375 380

atg?agc?ccc?acc?ttc?atc?aac?atg?tct?cag?gag?aat?gtg?gac?atg?agg 1199

Met?Ser?Pro?Thr?Phe?Ile?Asn?Met?Ser?Gln?Glu?Asn?Val?Asp?Met?Arg

385 390 395

gac?tct?tac?ctg?ggt?tac?tcc?acc?gag?cta?gcc?ctg?tgg?aag?ggg?gta 1247

Asp?Ser?Tyr?Leu?Gly?Tyr?Ser?Thr?Glu?Leu?Ala?Leu?Trp?Lys?Gly?Val

400 405 410 415

cag?aac?ctg?gtc?ctg?ggg?gcc?ccc?cgc?tac?cag?cat?acc?ggg?aag?gct 1295

Gln?Asn?Leu?Val?Leu?Gly?Ala?Pro?Arg?Tyr?Gln?His?Thr?Gly?Lys?Ala

420 425 430

gtc?atc?ttc?acc?cag?gtg?tcc?agg?caa?tgg?agg?aag?aag?gcc?gaa?gtc 1343

Val?Ile?Phe?Thr?Gln?Val?Ser?Arg?Gln?Trp?Arg?Lys?Lys?Ala?Glu?Val

435 440 445

aca?ggg?acg?cag?atc?ggc?tcc?tac?ttc?ggg?gcc?tcc?ctc?tgc?tcc?gtg 1391

Thr?Gly?Thr?Gln?Ile?Gly?Ser?Tyr?Phe?Gly?Ala?Ser?Leu?Cys?Ser?Val

450 455 460

gat?gtg?gac?agc?gat?ggc?agc?acc?gac?ctg?atc?ctc?att?ggg?gcc?ccc 1439

Asp?Val?Asp?Ser?Asp?Gly?Ser?Thr?Asp?Leu?Ile?Leu?Ile?Gly?Ala?Pro

465 470 475

cat?tac?tat?gag?cag?acc?cga?ggg?ggc?cag?gtg?tcc?gtg?tgt?ccc?ttg 1487

His?Tyr?Tyr?Glu?Gln?Thr?Arg?Gly?Gly?Gln?Val?Ser?Val?Cys?Pro?Leu

480 485 490 495

cct?agg?ggg?cag?agg?gtg?cag?tgg?cag?tgt?gac?gct?gtt?ctc?cgt?ggt 1535

Pro?Arg?Gly?Gln?Arg?Val?Gln?Trp?Gln?Cys?Asp?Ala?Val?Leu?Arg?Gly

500 505 510

gag?cag?ggc?cac?ccc?tgg?ggc?cgc?ttt?ggg?gca?gcc?ctg?aca?gtg?ttg 1583

Glu?Gln?Gly?His?Pro?Trp?Gly?Arg?Phe?Gly?Ala?Ala?Leu?Thr?Val?Leu

515 520 525

ggg?gat?gtg?aat?gag?gac?aag?ctg?ata?gac?gtg?gcc?att?ggg?gcc?ccg 1631

Gly?Asp?Val?Asn?Glu?Asp?Lys?Leu?Ile?Asp?Val?Ala?Ile?Gly?Ala?Pro

530 535 540

gga?gag?cag?gag?aac?cgg?ggt?gct?gtc?tac?ctg?ttt?cac?gga?gcc?tca 1679

Gly?Glu?Gln?Glu?Asn?Arg?Gly?Ala?Val?Tyr?Leu?Phe?His?Gly?Ala?Ser

545 550 555

gaa?tcc?ggc?atc?agc?ccc?tcc?cac?agc?cag?cgg?att?gcc?agc?tcc?cag 1727

Glu?Ser?Gly?Ile?Ser?Pro?Ser?His?Ser?Gln?Arg?Ile?Ala?Ser?Ser?Gln

560 565 570 575

ctc?tcc?ccc?agg?ctg?cag?tat?ttt?ggg?cag?gcg?ctg?agt?ggg?ggt?cag 1775

Leu?Ser?Pro?Arg?Leu?Gln?Tyr?Phe?Gly?Gln?Ala?Leu?Ser?Gly?Gly?Gln

580 585 590

gac?ctc?acc?cag?gat?gga?ctg?atg?gac?ctg?gcc?gtg?ggg?gcc?cgg?ggc 1823

Asp?Leu?Thr?Gln?Asp?Gly?Leu?Met?Asp?Leu?Ala?Val?Gly?Ala?Arg?Gly

595 600 605

cag?gtg?ctc?ctg?ctc?agg?agt?ctg?ccg?gtg?ctg?aaa?gtg?ggg?gtg?gcc 1871

Gln?Val?Leu?Leu?Leu?Arg?Ser?Leu?Pro?Val?Leu?Lys?Val?Gly?Val?Ala

610 615 620

atg?aga?ttc?agc?cct?gtg?gag?gtg?gcc?aag?gct?gtg?tac?cgg?tgc?tgg 1919

Met?Arg?Phe?Ser?Pro?Val?Glu?Val?Ala?Lys?Ala?Val?Tyr?Arg?Cys?Trp

625 630 635

gaa?gag?aag?ccc?agt?gcc?ctg?gaa?gct?ggg?gac?gcc?acc?gtc?tgt?ctc 1967

Glu?Glu?Lys?Pro?Ser?Ala?Leu?Glu?Ala?Gly?Asp?Ala?Thr?Val?Cys?Leu

640 645 650 655

acc?atc?cag?aaa?agc?tca?ctg?gac?cag?cta?ggt?gac?atc?caa?agc?tct 2015

Thr?Ile?Gln?Lys?Ser?Ser?Leu?Asp?Gln?Leu?Gly?Asp?Ile?Gln?Ser?Ser

660 665 670

gtc?agg?ttt?gat?ctg?gca?ctg?gac?cca?ggt?cgt?ctg?act?tct?cgt?gcc 2063

Val?Arg?Phe?Asp?Leu?Ala?Leu?Asp?Pro?Gly?Arg?Leu?Thr?Ser?Arg?Ala

675 680 685

att?ttc?aat?gaa?acc?aag?aac?ccc?act?ttg?act?cga?aga?aaa?acc?ctg 2111

Ile?Phe?Asn?Glu?Thr?Lys?Asn?Pro?Thr?Leu?Thr?Arg?Arg?Lys?Thr?Leu

690 695 700

gga?ctg?ggg?att?cac?tgt?gaa?acc?ctg?aag?ctg?ctt?ttg?cca?gat?tgt 2159

Gly?Leu?Gly?Ile?His?Cys?Glu?Thr?Leu?Lys?Leu?Leu?Leu?Pro?Asp?Cys

705 710 715

gtg?gag?gat?gtg?gtg?agc?ccc?atc?att?ctg?cac?ctc?aac?ttc?tca?ctg 2207

Val?Glu?Asp?Val?Val?Ser?Pro?Ile?Ile?Leu?His?Leu?Asn?Phe?Ser?Leu

720 725 730 735

gtg?aga?gag?ccc?atc?ccc?tcc?ccc?cag?aac?ctg?cgt?cct?gtg?ctg?gcc 2255

Val?Arg?Glu?Pro?Ile?Pro?Ser?Pro?Gln?Asn?Leu?Arg?Pro?Val?Leu?Ala

740 745 750

gtg?ggc?tca?caa?gac?ctc?ttc?act?gct?tct?ctc?ccc?ttc?gag?aag?aac 2303

Val?Gly?Ser?Gln?Asp?Leu?Phe?Thr?Ala?Ser?Leu?Pro?Phe?Glu?Lys?Asn

755 760 765

tgt?ggg?caa?gat?ggc?ctc?tgt?gaa?ggg?gac?ctg?ggt?gtc?acc?ctc?agc 2351

Cys?Gly?Gln?Asp?Gly?Leu?Cys?Glu?Gly?Asp?Leu?Gly?Val?Thr?Leu?Ser

770 775 780

ttc?tca?ggc?ctg?cag?acc?ctg?acc?gtg?ggg?agc?tcc?ctg?gag?ctc?aac 2399

Phe?Ser?Gly?Leu?Gln?Thr?Leu?Thr?Val?Gly?Ser?Ser?Leu?Glu?Leu?Asn

785 790 795

gtg?att?gtg?act?gtg?tgg?aac?gca?ggt?gag?gat?tcc?tac?gga?acc?gtg 2447

Val?Ile?Val?Thr?Val?Trp?Asn?Ala?Gly?Glu?Asp?Ser?Tyr?Gly?Thr?Val

800 805 810 815

gtc?agc?ctc?tac?tat?cca?gca?ggg?ctg?tcg?cac?cga?cgg?gtg?tca?gga 2495

Val?Ser?Leu?Tyr?Tyr?Pro?Ala?Gly?Leu?Ser?His?Arg?Arg?Val?Ser?Gly

820 825 830

gcc?cag?aag?cag?ccc?cat?cag?agt?gcc?ctg?cgc?ctg?gca?tgt?gag?aca 2543

Ala?Gln?Lys?Gln?Pro?His?Gln?Ser?Ala?Leu?Arg?Leu?Ala?Cys?Glu?Thr

835 840 845

gtg?ccc?act?gag?gat?gag?ggc?cta?aga?agc?agc?cgc?tgc?agt?gtc?aac 2591

Val?Pro?Thr?Glu?Asp?Glu?Gly?Leu?Arg?Ser?Ser?Arg?Cys?Ser?Val?Asn

850 855 860

cac?ccc?atc?ttc?cat?gag?ggc?tct?aac?ggc?acc?ttc?ata?gtc?aca?ttc 2639

His?Pro?Ile?Phe?His?Glu?Gly?Ser?Asn?Gly?Thr?Phe?Ile?Val?Thr?Phe

865 870 875

gat?gtc?tcc?tac?aag?gcc?acc?ctg?gga?gac?agg?atg?ctt?atg?agg?gcc 2687

Asp?Val?Ser?Tyr?Lys?Ala?Thr?Leu?Gly?Asp?Arg?Met?Leu?Met?Arg?Ala

880 885 890 895

agt?gca?agc?agt?gag?aac?aat?aag?gct?tca?agc?agc?aag?gcc?acc?ttc 2735

Ser?Ala?Ser?Ser?Glu?Asn?Asn?Lys?Ala?Ser?Ser?Ser?Lys?Ala?Thr?Phe

900 905 910

cag?ctg?gag?ctc?ccg?gtg?aag?tat?gca?gtc?tac?acc?atg?atc?agc?agg 2783

Gln?Leu?Glu?Leu?Pro?Val?Lys?Tyr?Ala?Val?Tyr?Thr?Met?Ile?Ser?Arg

915 920 925

cag?gaa?gaa?tcc?acc?aag?tac?ttc?aac?ttt?gca?acc?tcc?gat?gag?aag 2831

Gln?Glu?Glu?Ser?Thr?Lys?Tyr?Phe?Asn?Phe?Ala?Thr?Ser?Asp?Glu?Lys

930 935 940

aaa?atg?aaa?gag?gct?gag?cat?cga?tac?cgt?gtg?aat?aac?ctc?agc?cag 2879

Lys?Met?Lys?Glu?Ala?Glu?His?Arg?Tyr?Arg?Val?Asn?Asn?Leu?Ser?Gln

945 950 955

cga?gat?ctg?gcc?atc?agc?att?aac?ttc?tgg?gtt?cct?gtc?ctg?ctg?aac 2927

Arg?Asp?Leu?Ala?Ile?Ser?Ile?Asn?Phe?Trp?Val?Pro?Val?Leu?Leu?Asn

960 965 970 975

ggg?gtg?gct?gtg?tgg?gat?gtg?gtc?atg?gag?gcc?cca?tct?cag?agt?ctc 2975

Gly?Val?Ala?Val?Trp?Asp?Val?Val?Met?Glu?Ala?Pro?Ser?Gln?Ser?Leu

980 985 990

ccc?tgt?gtt?tca?gag?aga?aaa?cct?ccc?cag?cat?tct?gac?ttc?ctg?acc 3023

Pro?Cys?Val?Ser?Glu?Arg?Lys?Pro?Pro?Gln?His?Ser?Asp?Phe?Leu?Thr

995 1000 1005

cag?att?tca?aga?agt?ccc?atg?ctg?gac?tgc?tcc?att?gct?gac?tgc?ctg 3071

Gln?Ile?Ser?Arg?Ser?Pro?Met?Leu?Asp?Cys?Ser?Ile?Ala?Asp?Cys?Leu

1010 1015 1020

cag?ttc?cgc?tgt?gac?gtc?ccc?tcc?ttc?agc?gtc?cag?gag?gag?ctg?gat 3119

Gln?Phe?Arg?Cys?Asp?Val?Pro?Ser?Phe?Ser?Val?Gln?Glu?Glu?Leu?Asp

1025 1030 1035

ttc?acc?ctg?aag?ggc?aat?ctc?agt?ttc?ggc?tgg?gtc?cgc?gag?aca?ttg 3167

Phe?Thr?Leu?Lys?Gly?Asn?Leu?Ser?Phe?Gly?Trp?Val?Arg?Glu?Thr?Leu

1040 1045 1050 1055

cag?aag?aag?gtg?ttg?gtc?gtg?agt?gtg?gct?gaa?att?acg?ttc?gac?aca 3215

Gln?Lys?Lys?Val?Leu?Val?Val?Ser?Val?Ala?Glu?Ile?Thr?Phe?Asp?Thr

1060 1065 1070

tcc?gtg?tac?tcc?cag?ctt?cca?gga?cag?gag?gca?ttt?atg?aga?gct?cag 3263

Ser?Val?Tyr?Ser?Gln?Leu?Pro?Gly?Gln?Glu?Ala?Phe?Met?Arg?Ala?Gln

1075 1080 1085

atg?gag?atg?gtg?cta?gaa?gaa?gac?gag?gtc?tac?aat?gcc?att?ccc?atc 3311

Met?Glu?Met?Val?Leu?Glu?Glu?Asp?Glu?Val?Tyr?Asn?Ala?Ile?Pro?Ile

1090 1095 1100

atc?atg?ggc?agc?tct?gtg?ggg?gct?ctg?cta?ctg?ctg?gcg?ctc?atc?aca 3359

Ile?Met?Gly?Ser?Ser?Val?Gly?Ala?Leu?Leu?Leu?Leu?Ala?Leu?Ile?Thr

1105 1110 1115

gcc?aca?ctg?tac?aag?ctt?ggc?ttc?ttc?aaa?cgc?cac?tac?aag?gaa?atg 3407

Ala?Thr?Leu?Tyr?Lys?Leu?Gly?Phe?Phe?Lys?Arg?His?Tyr?Lys?Glu?Met

1120 1125 1130 1135

ctg?gag?gac?aag?cct?gaa?gac?act?gcc?aca?ttc?agt?ggg?gac?gat?ttc 3455

Leu?Glu?Asp?Lys?Pro?Glu?Asp?Thr?Ala?Thr?Phe?Ser?Gly?Asp?Asp?Phe

1140 1145 1150

agc?tgt?gtg?gcc?cca?aat?gtg?cct?ttg?tcc?taataatcca?ctttcctgtt 3505

Ser?Cys?Val?Ala?Pro?Asn?Val?Pro?Leu?Ser

1155 1160

tatctctacc?actgtgggct?ggacttgctt?gcaaccataa?atcaacttac?atggaaacaa?3565

cttctgcata?gatctgcact?ggcctaagca?acctaccagg?tgctaagcac?cttctcggag?3625

agatagagat?tgtaatgttt?ttacatatct?gtccatcttt?ttcagcaatg?acccactttt?3685

tacagaagca?ggcatggtgc?cagcataaat?tttcatatgc?t 3726

<210>2

<211>1161

<212>PRT

＜213〉homo sapiens

<400>2

Thr?Phe?Gly?Thr?Val?Leu?Leu?Leu?Ser?Val?Leu?Ala?Ser?Tyr?His?Gly

1 5 10 15

Phe?Asn?Leu?Asp?Val?Glu?Glu?Pro?Thr?Ile?Phe?Gln?Glu?Asp?Ala?Gly

20 25 30

Gly?Phe?Gly?Gln?Ser?Val?Val?Gln?Phe?Gly?Gly?Ser?Arg?Leu?Val?Val

35 40 45

Gly?Ala?Pro?Leu?Glu?Val?Val?Ala?Ala?Asn?Gln?Thr?Gly?Arg?Leu?Tyr

50 55 60

Asp?Cys?Ala?Ala?Ala?Thr?Gly?Met?Cys?Gln?Pro?Ile?Pro?Leu?His?Ile

65 70 75 80

Arg?Pro?Glu?Ala?Val?Asn?Met?Ser?Leu?Gly?Leu?Thr?Leu?Ala?Ala?Ser

85 90 95

Thr?Asn?Gly?Ser?Arg?Leu?Leu?Ala?Cys?Gly?Pro?Thr?Leu?His?Arg?Val

100 105 110

Cys?Gly?Glu?Asn?Ser?Tyr?Ser?Lys?Gly?Ser?Cys?Leu?Leu?Leu?Gly?Ser

115 120 125

Arg?Trp?Glu?Ile?Ile?Gln?Thr?Val?Pro?Asp?Ala?Thr?Pro?Glu?Cys?Pro

130 135 140

His?Gln?Glu?Met?Asp?Ile?Val?Phe?Leu?Ile?Asp?Gly?Ser?Gly?Ser?Ile

145 150 155 160

Asp?Gln?Asn?Asp?Phe?Asn?Gln?Met?Lys?Gly?Phe?Val?Gln?Ala?Val?Met

165 170 175

Gly?Gln?Phe?Glu?Gly?Thr?Asp?Thr?Leu?Phe?Ala?Leu?Met?Gln?Tyr?Ser

180 185 190

Asn?Leu?Leu?Lys?Ile?His?Phe?Thr?Phe?Thr?Gln?Phe?Arg?Thr?Ser?Pro

195 200 205

Ser?Gln?Gln?Ser?Leu?Val?Asp?Pro?Ile?Val?Gln?Leu?Lys?Gly?Leu?Thr

210 215 220

Phe?Thr?Ala?Thr?Gly?Ile?Leu?Thr?Val?Val?Thr?Gln?Leu?Phe?His?His

225 230 235 240

Lys?Asn?Gly?Ala?Arg?Lys?Ser?Ala?Lys?Lys?Ile?Leu?Ile?Val?Ile?Thr

245 250 255

Asp?Gly?Gln?Lys?Tyr?Lys?Asp?Pro?Leu?Glu?Tyr?Ser?Asp?Val?Ile?Pro

260 265 270

Gln?Ala?Glu?Lys?Ala?Gly?Ile?Ile?Arg?Tyr?Ala?Ile?Gly?Val?Gly?His

275 280 285

Ala?Phe?Gln?Gly?Pro?Thr?Ala?Arg?Gln?Glu?Leu?Asn?Thr?Ile?Ser?Ser

290 295 300

Ala?Pro?Pro?Gln?Asp?His?Val?Phe?Lys?Val?Asp?Asn?Phe?Ala?Ala?Leu

305 310 315 320

Gly?Ser?Ile?Gln?Lys?Gln?Leu?Gln?Glu?Lys?Ile?Tyr?Ala?Val?Glu?Gly

325 330 335

Thr?Gln?Ser?Arg?Ala?Ser?Ser?Ser?Phe?Gln?His?Glu?Met?Ser?Gln?Glu

340 345 350

Gly?Phe?Ser?Thr?Ala?Leu?Thr?Met?Asp?Gly?Leu?Phe?Leu?Gly?Ala?Val

355 360 365

Gly?Ser?Phe?Ser?Trp?Ser?Gly?Gly?Ala?Phe?Leu?Tyr?Pro?Pro?Asn?Met

370 375 380

Ser?Pro?Thr?Phe?Ile?Asn?Met?Ser?Gln?Glu?Asn?Val?Asp?Met?Arg?Asp

385 390 395 400

Ser?Tyr?Leu?Gly?Tyr?Ser?Thr?Glu?Leu?Ala?Leu?Trp?Lys?Gly?Val?Gln

405 410 415

Asn?Leu?Val?Leu?Gly?Ala?Pro?Arg?Tyr?Gln?His?Thr?Gly?Lys?Ala?Val

420 425 430

Ile?Phe?Thr?Gln?Val?Ser?Arg?Gln?Trp?Arg?Lys?Lys?Ala?Glu?Val?Thr

435 440 445

Gly?Thr?Gln?Ile?Gly?Ser?Tyr?Phe?Gly?Ala?Ser?Leu?Cys?Ser?Val?Asp

450 455 460

Val?Asp?Ser?Asp?Gly?Ser?Thr?Asp?Leu?Ile?Leu?Ile?Gly?Ala?Pro?His

465 470 475 480

Tyr?Tyr?Glu?Gln?Thr?Arg?Gly?Gly?Gln?Val?Ser?Val?Cys?Pro?Leu?Pro

485 490 495

Arg?Gly?Gln?Arg?Val?Gln?Trp?Gln?Cys?Asp?Ala?Val?Leu?Arg?Gly?Glu

500 505 510

Gln?Gly?His?Pro?Trp?Gly?Arg?Phe?Gly?Ala?Ala?Leu?Thr?Val?Leu?Gly

515 520 525

Asp?Val?Asn?Glu?Asp?Lys?Leu?Ile?Asp?Val?Ala?Ile?Gly?Ala?Pro?Gly

530 535 540

Glu?Gln?Glu?Asn?Arg?Gly?Ala?Val?Tyr?Leu?Phe?His?Gly?Ala?Ser?Glu

545 550 555 560

Ser?Gly?Ile?Ser?Pro?Ser?His?Ser?Gln?Arg?Ile?Ala?Ser?Ser?Gln?Leu

565 570 575

Ser?Pro?Arg?Leu?Gln?Tyr?Phe?Gly?Gln?Ala?Leu?Ser?Gly?Gly?Gln?Asp

580 585 590

Leu?Thr?Gln?Asp?Gly?Leu?Met?Asp?Leu?Ala?Val?Gly?Ala?Arg?Gly?Gln

595 600 605

Val?Leu?Leu?Leu?Arg?Ser?Leu?Pro?Val?Leu?Lys?Val?Gly?Val?Ala?Met

610 615 620

Arg?Phe?Ser?Pro?Val?Glu?Val?Ala?Lys?Ala?Val?Tyr?Arg?Cys?Trp?Glu

625 630 635 640

Glu?Lys?Pro?Ser?Ala?Leu?Glu?Ala?Gly?Asp?Ala?Thr?Val?Cys?Leu?Thr

645 650 655

Ile?Gln?Lys?Ser?Ser?Leu?Asp?Gln?Leu?Gly?Asp?Ile?Gln?Ser?Ser?Val

660 665 670

Arg?Phe?Asp?Leu?Ala?Leu?Asp?Pro?Gly?Arg?Leu?Thr?Ser?Arg?Ala?Ile

675 680 685

Phe?Asn?Glu?Thr?Lys?Asn?Pro?Thr?Leu?Thr?Arg?Arg?Lys?Thr?Leu?Gly

690 695 700

Leu?Gly?Ile?His?Cys?Glu?Thr?Leu?Lys?Leu?Leu?Leu?Pro?Asp?Cys?Val

705 710 715 720

Glu?Asp?Val?Val?Ser?Pro?Ile?Ile?Leu?His?Leu?Asn?Phe?Ser?Leu?Val

725 730 735

Arg?Glu?Pro?Ile?Pro?Ser?Pro?Gln?Asn?Leu?Arg?Pro?Val?Leu?Ala?Val

740 745 750

Gly?Ser?Gln?Asp?Leu?Phe?Thr?Ala?Ser?Leu?Pro?Phe?Glu?Lys?Asn?Cys

755 760 765

Gly?Gln?Asp?Gly?Leu?Cys?Glu?Gly?Asp?Leu?Gly?Val?Thr?Leu?Ser?Phe

770 775 780

Ser?Gly?Leu?Gln?Thr?Leu?Thr?Val?Gly?Ser?Ser?Leu?Glu?Leu?Asn?Val

785 790 795 800

Ile?Val?Thr?Val?Trp?Asn?Ala?Gly?Glu?Asp?Ser?Tyr?Gly?Thr?Val?Val

805 810 815

Ser?Leu?Tyr?Tyr?Pro?Ala?Gly?Leu?Ser?His?Arg?Arg?Val?Ser?Gly?Ala

820 825 830

Gln?Lys?Gln?Pro?His?Gln?Ser?Ala?Leu?Arg?Leu?Ala?Cys?Glu?Thr?Val

835 840 845

Pro?Thr?Glu?Asp?Glu?Gly?Leu?Arg?Ser?Ser?Arg?Cys?Ser?Val?Asn?His

850 855 860

Pro?Ile?Phe?His?Glu?Gly?Ser?Asn?Gly?Thr?Phe?Ile?Val?Thr?Phe?Asp

865 870 875 880

Val?Ser?Tyr?Lys?Ala?Thr?Leu?Gly?Asp?Arg?Met?Leu?Met?Arg?Ala?Ser

885 890 895

Ala?Ser?Ser?Glu?Asn?Asn?Lys?Ala?Ser?Ser?Ser?Lys?Ala?Thr?Phe?Gln

900 905 910

Leu?Glu?Leu?Pro?Val?Lys?Tyr?Ala?Val?Tyr?Thr?Met?Ile?Ser?Arg?Gln

915 920 925

Glu?Glu?Ser?Thr?Lys?Tyr?Phe?Asn?Phe?Ala?Thr?Ser?Asp?Glu?Lys?Lys

930 935 940

Met?Lys?Glu?Ala?Glu?His?Arg?Tyr?Arg?Val?Asn?Asn?Leu?Ser?Gln?Arg

945 950 955 960

Asp?Leu?Ala?Ile?Ser?Ile?Asn?Phe?Trp?Val?Pro?Val?Leu?Leu?Asn?Gly

965 970 975

Val?Ala?Val?Trp?Asp?Val?Val?Met?Glu?Ala?Pro?Ser?Gln?Ser?Leu?Pro

980 985 990

Cys?Val?Ser?Glu?Arg?Lys?Pro?Pro?Gln?His?Ser?Asp?Phe?Leu?Thr?Gln

995 1000 1005

Ile?Ser?Arg?Ser?Pro?Met?Leu?Asp?Cys?Ser?Ile?Ala?Asp?Cys?Leu?Gln

1010 1015 1020

Phe?Arg?Cys?Asp?Val?Pro?Ser?Phe?Ser?Val?Gln?Glu?Glu?Leu?Asp?Phe

1025 1030 1035 1040

Thr?Leu?Lys?Gly?Asn?Leu?Ser?Phe?Gly?Trp?Val?Arg?Glu?Thr?Leu?Gln

1045 1050 1055

Lys?Lys?Val?Leu?Val?Val?Ser?Val?Ala?Glu?Ile?Thr?Phe?Asp?Thr?Ser

1060 1065 1070

Val?Tyr?Ser?Gln?Leu?Pro?Gly?Gln?Glu?Ala?Phe?Met?Arg?Ala?Gln?Met

1075 1080 1085

Glu?Met?Val?Leu?Glu?Glu?Asp?Glu?Val?Tyr?Asn?Ala?Ile?Pro?Ile?Ile

1090 1095 1100

Met?Gly?Ser?Ser?Val?Gly?Ala?Leu?Leu?Leu?Leu?Ala?Leu?Ile?Thr?Ala

1105 1110 1115 1120

Thr?Leu?Tyr?Lys?Leu?Gly?Phe?Phe?Lys?Arg?His?Tyr?Lys?Glu?Met?Leu

1125 1130 1135

Glu?Asp?Lys?Pro?Glu?Asp?Thr?Ala?Thr?Phe?Ser?Gly?Asp?Asp?Phe?Ser

1140 1145 1150

Cys?Val?Ala?Pro?Asn?Val?Pro?Leu?Ser

1155 1160

<210>3

<211>1153

<212>PRT

＜213〉homo sapiens

<400>3

Met?Ala?Leu?Arg?Val?Leu?Leu?Leu?Thr?Ala?Leu?Thr?Leu?Cys?His?Gly

1 5 10 15

Phe?Asn?Leu?Asp?Thr?Glu?Asn?Ala?Met?Thr?Phe?Gln?Glu?Asn?Ala?Arg

20 25 30

Gly?Phe?Gly?Gln?Ser?Val?Val?Gln?Leu?Gln?Gly?Ser?Arg?Val?Val?Val

35 40 45

Gly?Ala?Pro?Gln?Glu?Ile?Val?Ala?Ala?Asn?Gln?Arg?Gly?Ser?Leu?Tyr

50 55 60

Gln?Cys?Asp?Tyr?Ser?Thr?Gly?Ser?Cys?Glu?Pro?Ile?Arg?Leu?Gln?Val

65 70 75 80

Pro?Val?Glu?Ala?Val?Asn?Met?Ser?Leu?Gly?Leu?Ser?Leu?Ala?Ala?Thr

85 90 95

Thr?Ser?Pro?Pro?Gln?Leu?Leu?Ala?Cys?Gly?Pro?Thr?Val?His?Gln?Thr

100 105 110

Cys?Ser?Glu?Asn?Thr?Tyr?Val?Lys?Gly?Leu?Cys?Phe?Leu?Phe?Gly?Ser

115 120 125

Asn?Leu?Arg?Gln?Gln?Pro?Gln?Lys?Phe?Pro?Glu?Ala?Leu?Arg?Gly?Cys

130 135 140

Pro?Gln?Glu?Asp?Ser?Asp?Ile?Ala?Phe?Leu?Ile?Asp?Gly?Ser?Gly?Ser

145 150 155 160

Ile?Ile?Pro?His?Asp?Phe?Arg?Arg?Met?Lys?Glu?Phe?Val?Ser?Thr?Val

165 170 175

Met?Glu?Gln?Leu?Lys?Lys?Ser?Lys?Thr?Leu?Phe?Ser?Leu?Met?Gln?Tyr

180 185 190

Ser?Glu?Glu?Phe?Arg?Ile?His?Phe?Thr?Phe?Lys?Glu?Phe?Gln?Asn?Asn

195 200 205

Pro?Asn?Pro?Arg?Ser?Leu?Val?Lys?Pro?Ile?Thr?Gln?Leu?Leu?Gly?Arg

210 215 220

Thr?His?Thr?Ala?Thr?Gly?Ile?Arg?Lys?Val?Val?Arg?Glu?Leu?Phe?Asn

225 230 235 240

Ile?Thr?Asn?Gly?Ala?Arg?Lys?Asn?Ala?Phe?Lys?Ile?Leu?Val?Val?Ile

245 250 255

Thr?Asp?Gly?Glu?Lys?Phe?Gly?Asp?Pro?Leu?Gly?Tyr?Glu?Asp?Val?Ile

260 265 270

Pro?Glu?Ala?Asp?Arg?Glu?Gly?Val?Ile?Arg?Tyr?Val?Ile?Gly?Val?Gly

275 280 285

Asp?Ala?Phe?Arg?Ser?Glu?Lys?Ser?Arg?Gln?Glu?Leu?Asn?Thr?Ile?Ala

290 295 300

Ser?Lys?Pro?Pro?Arg?Asp?His?Val?Phe?Gln?Val?Asn?Asn?Phe?Glu?Ala

305 310 315 320

Leu?Lys?Thr?Ile?Gln?Asn?Gln?Leu?Arg?Glu?Lys?Ile?Phe?Ala?Ile?Glu

325 330 335

Gly?Thr?Gln?Thr?Gly?Ser?Ser?Ser?Ser?Phe?Glu?His?Glu?Met?Ser?Gln

340 345 350

Glu?Gly?Phe?Ser?Ala?Ala?Ile?Thr?Ser?Asn?Gly?Pro?Leu?Leu?Ser?Thr

355 360 365

Val?Gly?Ser?Tyr?Asp?Trp?Ala?Gly?Gly?Val?Phe?Leu?Tyr?Thr?Ser?Lys

370 375 380

Glu?Lys?Ser?Thr?Phe?Ile?Asn?Met?Thr?Arg?Val?Asp?Ser?Asp?Met?Asn

385 390 395 400

Asp?Ala?Tyr?Leu?Gly?Tyr?Ala?Ala?Ala?Ile?Ile?Leu?Arg?Asn?Arg?Val

405 410 415

Gln?Ser?Leu?Val?Leu?Gly?Ala?Pro?Arg?Tyr?Gln?His?Ile?Gly?Leu?Val

420 425 430

Ala?Met?Phe?Arg?Gln?Asn?Thr?Gly?Met?Trp?Glu?Ser?Asn?Ala?Asn?Val

435 440 445

Lys?Gly?Thr?Gln?Ile?Gly?Ala?Tyr?Phe?Gly?Ala?Ser?Leu?Cys?Ser?Val

450 455 460

Asp?Val?Asp?Ser?Asn?Gly?Ser?Thr?Asp?Leu?Val?Leu?Ile?Gly?Ala?Pro

465 470 475 480

His?Tyr?Tyr?Glu?Gln?Thr?Arg?Gly?Gly?Gln?Val?Ser?Val?Cys?Pro?Leu

485 490 495

Pro?Arg?Gly?Gln?Arg?Ala?Arg?Trp?Gln?Cys?Asp?Ala?Val?Leu?Tyr?Gly

500 505 510

Glu?Gln?Gly?Gln?Pro?Trp?Gly?Arg?Phe?Gly?Ala?Ala?Leu?Thr?Val?Leu

515 520 525

Gly?Asp?Val?Asn?Gly?Asp?Lys?Leu?Thr?Asp?Val?Ala?Ile?Gly?Ala?Pro

530 535 540

Gly?Glu?Glu?Asp?Asn?Arg?Gly?Ala?Val?Tyr?Leu?Phe?His?Gly?Thr?Ser

545 550 555 560

Gly?Ser?Gly?Ile?Ser?Pro?Ser?His?Ser?Gln?Arg?Ile?Ala?Gly?Ser?Lys

565 570 575

Leu?Ser?Pro?Arg?Leu?Gln?Tyr?Phe?Gly?Gln?Ser?Leu?Ser?Gly?Gly?Gln

580 585 590

Asp?Leu?Thr?Met?Asp?Gly?Leu?Val?Asp?Leu?Thr?Val?Gly?Ala?Gln?Gly

595 600 605

His?Val?Leu?Leu?Leu?Arg?Ser?Gln?Pro?Val?Leu?Arg?Val?Lys?Ala?Ile

610 615 620

Met?Glu?Phe?Asn?Pro?Arg?Glu?Val?Ala?Arg?Asn?Val?Phe?Glu?Cys?Asn

625 630 635 640

Asp?Gln?Val?Val?Lys?Gly?Lys?Glu?Ala?Gly?Glu?Val?Arg?Val?Cys?Leu

645 650 655

His?Val?Gln?Lys?Ser?Thr?Arg?Asp?Arg?Leu?Arg?Glu?Gly?Gln?Ile?Gln

660 665 670

Ser?Val?Val?Thr?Tyr?Asp?Leu?Ala?Leu?Asp?Ser?Gly?Arg?Pro?His?Ser

675 680 685

Arg?Ala?Val?Phe?Asn?Glu?Thr?Lys?Asn?Ser?Thr?Arg?Arg?Gln?Thr?Gln

690 695 700

Val?Leu?Gly?Leu?Thr?Gln?Thr?Cys?Glu?Thr?Leu?Lys?Leu?Gln?Leu?Pro

705 710 715 720

Asn?Cys?Ile?Glu?Asp?Pro?Val?Ser?Pro?Ile?Val?Leu?Arg?Leu?Asn?Phe

725 730 735

Ser?Leu?Val?Gly?Thr?Pro?Leu?Ser?Ala?Phe?Gly?Asn?Leu?Arg?Pro?Val

740 745 750

Leu?Ala?Glu?Asp?Ala?Gln?Arg?Leu?Phe?Thr?Ala?Leu?Phe?Pro?Phe?Glu

755 760 765

Lys?Asn?Cys?Gly?Asn?Asp?Asn?Ile?Cys?Gln?Asp?Asp?Leu?Ser?Ile?Thr

770 775 780

Phe?Ser?Phe?Met?Ser?Leu?Asp?Cys?Leu?Val?Val?Gly?Gly?Pro?Arg?Glu

785 790 795 800

Phe?Asn?Val?Thr?Val?Thr?Val?Arg?Asn?Asp?Gly?Glu?Asp?Ser?Tyr?Arg

805 810 815

Thr?Gln?Val?Thr?Phe?Phe?Phe?Pro?Leu?Asp?Leu?Ser?Tyr?Arg?Lys?Val

820 825 830

Ser?Thr?Leu?Gln?Asn?Gln?Arg?Ser?Gln?Arg?Ser?Trp?Arg?Leu?Ala?Cys

835 840 845

Glu?Ser?Ala?Ser?Ser?Thr?Glu?Val?Ser?Gly?Ala?Leu?Lys?Ser?Thr?Ser

850 855 860

Cys?Ser?Ile?Asn?His?Pro?Ile?Phe?Pro?Glu?Asn?Ser?Glu?Val?Thr?Phe

865 870 875 880

Asn?Ile?Thr?Phe?Asp?Val?Asp?Ser?Lys?Ala?Ser?Leu?Gly?Asn?Lys?Leu

885 890 895

Leu?Leu?Lys?Ala?Asn?Val?Thr?Ser?Glu?Asn?Asn?Met?Pro?Arg?Thr?Asn

900 905 910

Lys?Thr?Glu?Phe?Gln?Leu?Glu?Leu?Pro?Val?Lys?Tyr?Ala?Val?Tyr?Met

915 920 925

Val?Val?Thr?Ser?His?Gly?Val?Ser?Thr?Lys?Tyr?Leu?Asn?Phe?Thr?Ala

930 935 940

Ser?Glu?Asn?Thr?Ser?Arg?Val?Met?Gln?His?Gln?Tyr?Gln?Val?Ser?Asn

945 950 955 960

Leu?Gly?Gln?Arg?Ser?Leu?Pro?Ile?Ser?Leu?Val?Phe?Leu?Val?Pro?Val

965 970 975

Arg?Leu?Asn?Gln?Thr?Val?Ile?Trp?Asp?Arg?Pro?Gln?Val?Thr?Phe?Ser

980 985 990

Glu?Asn?Leu?Ser?Ser?Thr?Cys?His?Thr?Lys?Glu?Arg?Leu?Pro?Ser?His

995 1000 1005

Ser?Asp?Phe?Leu?Ala?Glu?Leu?Arg?Lys?Ala?Pro?Val?Val?Asn?Cys?Ser

1010 1015 1020

Ile?Ala?Val?Cys?Gln?Arg?Ile?Gln?Cys?Asp?Ile?Pro?Phe?Phe?Gly?Ile

1025 1030 1035 1040

Gln?Glu?Glu?Phe?Asn?Ala?Thr?Leu?Lys?Gly?Asn?Leu?Ser?Phe?Asp?Trp

1045 1050 1055

Tyr?Ile?Lys?Thr?Ser?His?Asn?His?Leu?Leu?Ile?Val?Ser?Thr?Ala?Glu

1060 1065 1070

Ile?Leu?Phe?Asn?Asp?Ser?Val?Phe?Thr?Leu?Leu?Pro?Gly?Gln?Gly?Ala

1075 1080 1085

Phe?Val?Arg?Ser?Gln?Thr?Glu?Thr?Lys?Val?Glu?Pro?Phe?Glu?Val?Pro

1090 1095 1100

Asn?Pro?Leu?Pro?Leu?Ile?Val?Gly?Ser?Ser?Val?Gly?Gly?Leu?Leu?Leu

1105 1110 1115 1120

Leu?Ala?Leu?Ile?Thr?Ala?Ala?Leu?Tyr?Lys?Leu?Gly?Phe?Phe?Lys?Arg

1125 1130 1135

Gln?Tyr?Lys?Asp?Met?Met?Ser?Glu?Gly?Gly?Pro?Pro?Gly?Ala?Glu?Pro

1140 1145 1150

Gln

<210>4

<211>1163

<212>PRT

＜213〉homo sapiens

<400>4

Met?Thr?Arg?Thr?Arg?Ala?Ala?Leu?Leu?Leu?Phe?Thr?Ala?Leu?Ala?Thr

1 5 10 15

Ser?Leu?Gly?Phe?Asn?Leu?Asp?Thr?Glu?Glu?Leu?Thr?Ala?Phe?Arg?Val

20 25 30

Asp?Ser?Ala?Gly?Phe?Gly?Asp?Ser?Val?Val?Gln?Tyr?Ala?Asn?Ser?Trp

35 40 45

Val?Val?Val?Gly?Ala?Pro?Gln?Lys?Ile?Ile?Ala?Ala?Asn?Gln?Ile?Gly

50 55 60

Gly?Leu?Tyr?Gln?Cys?Gly?Tyr?Ser?Thr?Gly?Ala?Cys?Glu?Pro?Ile?Gly

65 70 75 80

Leu?Gln?Val?Pro?Pro?Glu?Ala?Val?Asn?Met?Ser?Leu?Gly?Leu?Ser?Leu

85 90 95

Ala?Ser?Thr?Thr?Ser?Pro?Ser?Gln?Leu?Leu?Ala?Cys?Gly?Pro?Thr?Val

100 105 110

His?His?Glu?Cys?Gly?Arg?Asn?Met?Tyr?Leu?Thr?Gly?Leu?Cys?Phe?Leu

115 120 125

Leu?Gly?Pro?Thr?Gln?Leu?Thr?Gln?Arg?Leu?Pro?Val?Ser?Arg?Gln?Glu

130 135 140

Cys?Pro?Arg?Gln?Glu?Gln?Asp?Ile?Val?Phe?Leu?Ile?Asp?Gly?Ser?Gly

145 150 155 160

Ser?Ile?Ser?Ser?Arg?Asn?Phe?Ala?Thr?Met?Met?Asn?Phe?Val?Arg?Ala

165 170 175

Val?Ile?Ser?Gln?Phe?Gln?Arg?Pro?Ser?Thr?Gln?Phe?Ser?Leu?Met?Gln

180 185 190

Phe?Ser?Asn?Lys?Phe?Gln?Thr?His?Phe?Thr?Phe?Glu?Glu?Phe?Arg?Arg

195 200 205

Thr?Ser?Asn?Pro?Leu?Ser?Leu?Leu?Ala?Ser?Val?His?Gln?Leu?Gln?Gly

210 215 220

Phe?Thr?Tyr?Thr?Ala?Thr?Ala?Ile?Gln?Asn?Val?Val?His?Arg?Leu?Phe

225 230 235 240

His?Ala?Ser?Tyr?Gly?Ala?Arg?Arg?Asp?Ala?Ile?Lys?Ile?Leu?Ile?Val

245 250 255

Ile?Thr?Asp?Gly?Lys?Lys?Glu?Gly?Asp?Ser?Leu?Asp?Tyr?Lys?Asp?Val

260 265 270

Ile?Pro?Met?Ala?Asp?Ala?Ala?Gly?Ile?Ile?Arg?Tyr?Ala?Ile?Gly?Val

275 280 285

Gly?Leu?Ala?Phe?Gln?Asn?Arg?Asn?Ser?Trp?Lys?Glu?Leu?Asn?Asp?Ile

290 295 300

Ala?Ser?Lys?Pro?Ser?Gln?Glu?His?Ile?Phe?Lys?Val?Glu?Asp?Phe?Asp

305 310 315 320

Ala?Leu?Lys?Asp?Ile?Gln?Asn?Gln?Leu?Lys?Glu?Lys?Ile?Phe?Ala?Ile

325 330 335

Glu?Gly?Thr?Glu?Thr?Ile?Ser?Ser?Ser?Ser?Phe?Glu?Leu?Glu?Met?Ala

340 345 350

Gln?Glu?Gly?Phe?Ser?Ala?Val?Phe?Thr?Pro?Asp?Gly?Pro?Val?Leu?Gly

355 360 365

Ala?Val?Gly?Ser?Phe?Thr?Trp?Ser?Gly?Gly?Ala?Phe?Leu?Tyr?Pro?Pro

370 375 380

Asn?Met?Ser?Pro?Thr?Phe?Ile?Asn?Met?Ser?Gln?Glu?Asn?Val?Asp?Met

385 390 395 400

Arg?Asp?Ser?Tyr?Leu?Gly?Tyr?Ser?Thr?Glu?Leu?Ala?Leu?Trp?Lys?Gly

405 410 415

Val?Gln?Ser?Leu?Val?Leu?Gly?Ala?Pro?Arg?Tyr?Gln?His?Ile?Gly?Lys

420 425 430

Ala?Val?Ile?Phe?Ile?Gln?Val?Ser?Arg?Gln?Trp?Arg?Met?Lys?Ala?Glu

435 440 445

Val?Ile?Gly?Thr?Gln?Ile?Gly?Ser?Tyr?Phe?Gly?Ala?Ser?Leu?Cys?Ser

450 455 460

Val?Asp?Val?Asp?Thr?Asp?Gly?Ser?Thr?Asp?Leu?Val?Leu?Ile?Gly?Ala

465 470 475 480

Pro?His?Tyr?Tyr?Glu?Gln?Thr?Arg?Gly?Gly?Gln?Val?Ser?Val?Cys?Pro

485 490 495

Leu?Pro?Arg?Gly?Trp?Arg?Arg?Trp?Trp?Cys?Asp?Ala?Val?Leu?Tyr?Gly

500 505 510

Glu?Gln?Gly?His?Pro?Trp?Gly?Arg?Phe?Gly?Ala?Ala?Leu?Thr?Val?Leu

515 520 525

Gly?Asp?Val?Asn?Gly?Asp?Lys?Leu?Thr?Asp?Val?Val?Ile?Gly?Ala?Pro

530 535 540

Gly?Glu?Glu?Glu?Asn?Arg?Gly?Ala?Val?Tyr?Leu?Phe?His?Gly?Val?Leu

545 550 555 560

Gly?Pro?Ser?Ile?Ser?Pro?Ser?His?Ser?Gln?Arg?Ile?Ala?Gly?Ser?Gln

565 570 575

Leu?Ser?Ser?Arg?Leu?Gln?Tyr?Phe?Gly?Gln?Ala?Leu?Ser?Gly?Gly?Gln

580 585 590

Asp?Leu?Thr?Gln?Asp?Gly?Leu?Val?Asp?Leu?Ala?Val?Gly?Ala?Arg?Gly

595 600 605

Gln?Val?Leu?Leu?Leu?Arg?Thr?Arg?Pro?Val?Leu?Trp?Val?Gly?Val?Ser

610 615 620

Met?Gln?Phe?Ile?Pro?Ala?Glu?Ile?Pro?Arg?Ser?Ala?Phe?Glu?Cys?Arg

625 630 635 640

Glu?Gln?Val?Val?Ser?Glu?Gln?Thr?Leu?Val?Gln?Ser?Asn?Ile?Cys?Leu

645 650 655

Tyr?Ile?Asp?Lys?Arg?Ser?Lys?Asn?Leu?Leu?Gly?Ser?Arg?Asp?Leu?Gln

660 665 670

Ser?Ser?Val?Thr?Leu?Asp?Leu?Ala?Leu?Ala?Pro?Gly?Arg?Leu?Ser?Pro

675 680 685

Arg?Ala?Ile?Phe?Gln?Glu?Thr?Lys?Asn?Arg?Ser?Leu?Ser?Arg?Val?Arg

690 695 700

Val?Leu?Gly?Leu?Lys?Ala?His?Cys?Glu?Asn?Phe?Asn?Leu?Leu?Leu?Pro

705 710 715 720

Ser?Cys?Val?Glu?Asp?Ser?Val?Ile?Pro?Ile?Ile?Leu?Arg?Leu?Asn?Phe

725 730 735

Thr?Leu?Val?Gly?Lys?Pro?Leu?Leu?Ala?Phe?Arg?Asn?Leu?Arg?Pro?Met

740 745 750

Leu?Ala?Ala?Leu?Ala?Gln?Arg?Tyr?Phe?Thr?Ala?Ser?Leu?Pro?Phe?Glu

755 760 765

Lys?Asn?Cys?Gly?Ala?Asp?His?Ile?Cys?Gln?Asp?Asn?Leu?Gly?Ile?Ser

770 775 780

Phe?Ser?Phe?Pro?Gly?Leu?Lys?Ser?Leu?Leu?Val?Gly?Ser?Asn?Leu?Glu

785 790 795 800

Leu?Asn?Ala?Glu?Val?Met?Val?Trp?Asn?Asp?Gly?Glu?Asp?Ser?Tyr?Gly

805 810 815

Thr?Thr?Ile?Thr?Phe?Ser?His?Pro?Ala?Gly?Leu?Ser?Tyr?Arg?Tyr?Val

820 825 830

Ala?Glu?Gly?Gln?Lys?Gln?Gly?Gln?Leu?Arg?Ser?Leu?His?Leu?Thr?Cys

835 840 845

Cys?Ser?Ala?Pro?Val?Gly?Ser?Gln?Gly?Thr?Trp?Ser?Thr?Ser?Cys?Arg

850 855 860

Ile?Asn?His?Leu?Ile?Phe?Arg?Gly?Gly?Ala?Gln?Ile?Thr?Phe?Leu?Ala

865 870 875 880

Thr?Phe?Asp?Val?Ser?Pro?Lys?Ala?Val?Gly?Leu?Asp?Arg?Leu?Leu?Leu

885 890 895

Ile?Ala?Asn?Val?Ser?Ser?Glu?Asn?Asn?Ile?Pro?Arg?Thr?Ser?Lys?Thr

900 905 910

Ile?Phe?Gln?Leu?Glu?Leu?Pro?Val?Lys?Tyr?Ala?Val?Tyr?Ile?Val?Val

915 920 925

Ser?Ser?His?Glu?Gln?Phe?Thr?Lys?Tyr?Leu?Asn?Phe?Ser?Glu?Ser?Glu

930 935 940

Glu?Lys?Glu?Ser?His?Val?Ala?Met?His?Arg?Tyr?Gln?Val?Asn?Asn?Leu

945 950 955 960

Gly?Gln?Arg?Asp?Leu?Pro?Val?Ser?Ile?Asn?Phe?Trp?Val?Pro?Val?Glu

965 970 975

Leu?Asn?Gln?Glu?Ala?Val?Trp?Met?Asp?Val?Glu?Val?Ser?His?Pro?Gln

980 985 990

Asn?Pro?Ser?Leu?Arg?Cys?Ser?Ser?Glu?Lys?Ile?Ala?Pro?Pro?Ala?Ser

995 1000 1005

Asp?Phe?Leu?Ala?His?Ile?Gln?Lys?Asn?Pro?Val?Leu?Asp?Cys?Ser?Ile

1010 1015 1020

Ala?Gly?Cys?Leu?Arg?Phe?Arg?Cys?Asp?Val?Pro?Ser?Phe?Ser?Val?Gln

1025 1030 1035 1040

Glu?Glu?Leu?Asp?Phe?Thr?Leu?Lys?Gly?Asn?Leu?Ser?Phe?Gly?Trp?Val

1045 1050 1055

Arg?Gln?Ile?Leu?Gln?Lys?Lys?Val?Ser?Val?Val?Ser?Val?Ala?Glu?Ile

1060 1065 1070

Ile?Phe?Asp?Thr?Ser?Val?Tyr?Ser?Gln?Leu?Pro?Gly?Gln?Glu?Ala?Phe

1075 1080 1085

Met?Arg?Ala?Gln?Thr?Ile?Thr?Val?Leu?Glu?Lys?Tyr?Lys?Val?His?Asn

1090 1095 1100

Pro?Ile?Pro?Leu?Ile?Val?Gly?Ser?Ser?Ile?Gly?Gly?Leu?Leu?Leu?Leu

1105 1110 1115 1120

Ala?Leu?Ile?Thr?Ala?Val?Leu?Tyr?Lys?Val?Gly?Phe?Phe?Lys?Arg?Gln

1125 1130 1135

Tyr?Lys?Glu?Met?Met?Glu?Glu?Ala?Asn?Gly?Gln?Ile?Ala?Pro?Glu?Asn

1140 1145 1150

Gly?Thr?Gln?Thr?Pro?Ser?Pro?Pro?Ser?Glu?Lys

1155 1160

<210>5

<211>12

<212>PRT

＜213〉Canis familiaris L.

<400>5

Phe?Asn?Leu?Asp?Val?Glu?Glu?Pro?Met?Val?Phe?Gln

1 5 10

<210>6

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>6

ttyaayytgg?aygtngarga?rccnatggtn?ttyca 35

<210>7

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>7

ttcaacctgg?acgtggagga?gcccatggtg?ttccaa 36

<210>8

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>8

ttcaacctgg?acgtngaasa?ncccatggtc?ttccaa 36

<210>9

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>9

ttyaayytng?aygtngarga?rcc 23

<210>10

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>10

ttyaayytgg?acgtngaaga 20

<210>11

<211>17

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>11

tgraanacca?tnggytc 17

<210>12

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>12

ttggaagacc?atnggytc 18

<210>13

<211>17

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>13

attaaccctc?actaaag 17

<210>14

<211>17

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>14

aatacgactc?actatag 17

<210>15

<211>11

<212>PRT

＜213〉Canis familiaris L.

<400>15

Val?Phe?Gln?Glu?Xaa?Gly?Ala?Gly?Phe?Gly?Gln

1 5 10

<210>16

<211>14

<212>PRT

＜213〉Canis familiaris L.

<400>16

Leu?Tyr?Asp?Xaa?Val?Ala?Ala?Thr?Gly?Leu?Xaa?Gln?Pro?Ile

1 5 10

<210>17

<211>12

<212>PRT

＜213〉Canis familiaris L.

<400>17

Pro?Leu?Glu?Tyr?Xaa?Asp?Val?Ile?Pro?Gln?Ala?Glu

1 5 10

<210>18

<211>10

<212>PRT

＜213〉Canis familiaris L.

<400>18

Phe?Gln?Glu?Gly?Phe?Ser?Xaa?Val?Leu?Xaa

1 5 10

<210>19

<211>14

<212>PRT

＜213〉Canis familiaris L.

<400>19

Thr?Ser?Pro?Thr?Phe?Ile?Xaa?Met?Ser?Gln?Glu?Asn?Val?Asp

1 5 10

<210>20

<211>17

<212>PRT

＜213〉Canis familiaris L.

<400>20

Leu?Val?Val?Gly?Ala?Pro?Leu?Glu?Val?Val?Ala?Val?Xaa?Gln?Thr?Gly

1 5 10 15

Arg

<210>21

<211>9

<212>PRT

＜213〉Canis familiaris L.

<400>21

Leu?Asp?Xaa?Lys?Pro?Xaa?Asp?Thr?Ala

1 5

<210>22

<211>7

<212>PRT

＜213〉Canis familiaris L.

<400>22

Phe?Gly?Glu?Gln?Phe?Ser?Glu

1 5

<210>23

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>23

raanccytcy?tgraaactyt?c 21

<210>24

<211>1006

<212>DNA

＜213〉Canis familiaris L.

<400>24

ttcaacctgg?acgtggagga?gcccatggtg?ttcaagagga?tggagctggc?tttggacaga?60

gcgtggccca?gcttggcgga?tctagactcg?tggtgggagc?ccccctggag?gtggtggcgg?120

tcaaccaaac?aggaaggttg?tatgactgtg?tggctgccac?tggccttgtc?aacccatacc?180

cctgcacaca?cccccagatg?ctgtgaacat?gtccctgggt?ctgtccctgt?cagccgccgc?240

cagtcgcccc?tggctgctgg?cctgtggccc?aaccatgcac?agagcctgtg?gggagaatat?300

gtatgcagaa?ggcttttgcc?tcctgttgga?ctcccatctg?cagaccattt?ggacagtacc?360

tgctgcccta?ccagagtgtc?caagtcaaga?gatggacatt?gtcttcctga?ttgatggttc?420

tggcagtatg?agcaaa?tga?ctttaaacaa?atgaaggatt?tgtgagagct?gtgatgggac?480

agtttgaggg?cacccaaacc?ctgttctcac?tgatacagta?tcccacctcc?ctgaagatcc?540

acttcacctt?cacgcaattc?cagagcagct?ggaaccctct?gagcctggtg?gatcccattg?600

tccaactgga?cggcctgaca?tatacagcca?cgggcatccg?gaaagtggtg?gaggaactgt?660

ttcatagtaa?gaatggggcc?cgtaaaagtg?ccaagaagat?cctcattgtc?atcacagatg?720

gcaaaaatac?aaagaccccc?tggagtacga?ggacgtatcc?ccaggcagag?agagcggatc?780

atccgctatg?ccattggggt?gggagatgct?ttctggaaac?ccagtgccaa?gcaggagctg?840

gacaaGattg?gctcagagcc?ggctcaggac?catgtgttca?gggtggacaa?ctttgcagca?900

ctcagcagca?tccaggagca?gctgcaggag?aagatctttg?cactcgaagg?aacccagtcg?960

acgacaagta?gctctttcca?acatgagatg?ttccaagaag?ggttca 1006

<210>25

<211>17

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>25

gtnttycarg?argaygg 17

<210>26

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>26

ccactgtcag?gatgcccgtg 20

<210>27

<211>42

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>27

agttacgaat?tcgccaccat?ggctctacgg?gtgcttcttc?tg 42

<210>28

<211>42

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>28

agttacgaat?tcgccaccat?gactcggact?gtgcttcttc?tg 42

<210>29

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>29

agttacgaat?tcgccaccat?gaccttcggc?actgtg 36

<210>30

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>30

ttgctgactg?cctgcagttc 20

<210>31

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>31

gttctgacgc?gtaatggcat?tgtagacctc?gtcttc 36

<210>32

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>32

acgtatgcag?gatcccatca?agagatggac?atcgct 36

<210>33

<211>37

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>33

actgcatgtc?tcgaggctga?agccttcttg?ggacatc 37

<210>34

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>34

tatagactgc?tgggtagtcc?ccac 24

<210>35

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>35

tgaagattgg?gggtaaataa?caga 24

<210>36

<211>3528

<212>DNA

＜213〉rat

<220>

<221>CDS

<222>(1)..(3453)

<220>

＜223〉artificial sequence note: primer

<400>36

ggc?tgg?gcc?ctg?gct?tcc?tgt?cat?ggg?tct?aac?ctg?gat?gtg?gag?gaa 48

Gly?Trp?Ala?Leu?Ala?Ser?Cys?His?Gly?Ser?Asn?Leu?Asp?Val?Glu?Glu

1 5 10 15

ccc?atc?gtg?ttc?aga?gag?gat?gca?gcc?agc?ttt?gga?cag?act?gtg?gtg 96

Pro?Ile?Val?Phe?Arg?Glu?Asp?Ala?Ala?Ser?Phe?Gly?Gln?Thr?Val?Val

20 25 30

cag?ttt?ggt?gga?tct?cga?ctc?gtg?gtg?gga?gcc?cct?ctg?gag?gcg?gtg 144

Gln?Phe?Gly?Gly?Ser?Arg?Leu?Val?Val?Gly?Ala?Pro?Leu?Glu?Ala?Val

35 40 45

gca?gtc?aac?caa?aca?gga?cgg?ttg?tat?gac?tgt?gca?cct?gcc?act?ggc 192

Ala?Val?Asn?Gln?Thr?Gly?Arg?Leu?Tyr?Asp?Cys?Ala?Pro?Ala?Thr?Gly

50 55 60

atg?tgc?cag?ccc?atc?gta?ctg?cgc?agt?ccc?cta?gag?gca?gtg?aac?atg 240

Met?Cys?Gln?Pro?Ile?Val?Leu?Arg?Ser?Pro?Leu?Glu?Ala?Val?Asn?Met

65 70 75 80

tcc?ctg?ggc?ctg?tct?ctg?gtg?act?gcc?acc?aat?aac?gcc?cag?ttg?ctg 288

Ser?Leu?Gly?Leu?Ser?Leu?Val?Thr?Ala?Thr?Asn?Asn?Ala?Gln?Leu?Leu

85 90 95

gct?tgt?ggt?cca?act?gca?cag?aga?gct?tgt?gtg?aag?aac?atg?tat?gcg 336

Ala?Cys?Gly?Pro?Thr?Ala?Gln?Arg?Ala?Cys?Val?Lys?Asn?Met?Tyr?Ala

100 105 110

aaa?ggt?tcc?tgc?ctc?ctt?ctc?ggc?tcc?agc?ttg?cag?ttc?atc?cag?gca 384

Lys?Gly?Ser?Cys?Leu?Leu?Leu?Gly?Ser?Ser?Leu?Gln?Phe?Ile?Gln?Ala

115 120 125

gtc?cct?gcc?tcc?atg?cca?gag?tgt?cca?aga?caa?gag?atg?gac?att?gct 432

Val?Pro?Ala?Ser?Met?Pro?Glu?Cys?Pro?Arg?Gln?Glu?Met?Asp?Ile?Ala

130 135 140

ttc?ctg?att?gat?ggt?tct?ggc?agc?att?aac?caa?agg?gac?ttt?gcc?cag 480

Phe?Leu?Ile?Asp?Gly?Ser?Gly?Ser?Ile?Asn?Gln?Arg?Asp?Phe?Ala?Gln

145 150 155 160

atg?aag?gac?ttt?gtc?aaa?gct?ttg?atg?gga?gag?ttt?gcg?agc?acc?agc 528

Met?Lys?Asp?Phe?Val?Lys?Ala?Leu?Met?Gly?Glu?Phe?Ala?Ser?Thr?Ser

165 170 175

acc?ttg?ttc?tcc?ctg?atg?caa?tac?tcg?aac?atc?ctg?aag?acc?cat?ttt 576

Thr?Leu?Phe?Ser?Leu?Met?Gln?Tyr?Ser?Asn?Ile?Leu?Lys?Thr?His?Phe

180 185 190

acc?ttc?act?gaa?ttc?aag?aac?atc?ctg?gac?cct?cag?agc?ctg?gtg?gat 624

Thr?Phe?Thr?Glu?Phe?Lys?Asn?Ile?Leu?Asp?Pro?Gln?Ser?Leu?Val?Asp

195 200 205

ccc?att?gtc?cag?ctg?caa?ggc?ctg?acc?tac?aca?gcc?aca?ggc?atc?cgg 672

Pro?Ile?Val?Gln?Leu?Gln?Gly?Leu?Thr?Tyr?Thr?Ala?Thr?Gly?Ile?Arg

210 215 220

aca?gtg?atg?gaa?gag?cta?ttt?cat?agc?aag?aat?ggg?tcc?cgt?aaa?agt 720

Thr?Val?Met?Glu?Glu?Leu?Phe?His?Ser?Lys?Asn?Gly?Ser?Arg?Lys?Ser

225 230 235 240

gcc?aag?aag?atc?ctc?ctt?gtc?atc?aca?gat?ggg?cag?aaa?tac?aga?gac 768

Ala?Lys?Lys?Ile?Leu?Leu?Val?Ile?Thr?Asp?Gly?Gln?Lys?Tyr?Arg?Asp

245 250 255

ccc?ctg?gag?tat?agt?gat?gtc?att?ccc?gcc?gca?gac?aaa?gct?ggc?atc 816

Pro?Leu?Glu?Tyr?Ser?Asp?Val?Ile?Pro?Ala?Ala?Asp?Lys?Ala?Gly?Ile

260 265 270

att?cgt?tat?gct?att?ggg?gtg?gga?gat?gcc?ttc?cag?gag?ccc?act?gcc 864

Ile?Arg?Tyr?Ala?Ile?Gly?Val?Gly?Asp?Ala?Phe?Gln?Glu?Pro?Thr?Ala

275 280 285

ctg?aag?gag?ctg?aac?acc?att?ggc?tca?gct?ccc?cca?cag?gac?cac?gtg 912

Leu?Lys?Glu?Leu?Asn?Thr?Ile?Gly?Ser?Ala?Pro?Pro?Gln?Asp?His?Val

290 295 300

ttc?aag?gta?ggc?aac?ttt?gca?gca?ctt?cgc?agc?atc?cag?agg?caa?ctt 960

Phe?Lys?Val?Gly?Asn?Phe?Ala?Ala?Leu?Arg?Ser?Ile?Gln?Arg?Gln?Leu

305 310 315 320

cag?gag?aaa?atc?ttc?gcc?att?gag?gga?act?caa?tca?agg?tca?agt?agt 1008

Gln?Glu?Lys?Ile?Phe?Ala?Ile?Glu?Gly?Thr?Gln?Ser?Arg?Ser?Ser?Ser

325 330 335

tcc?ttt?cag?cac?gag?atg?tca?caa?gaa?ggt?ttc?agt?tca?gct?ctc?aca 1056

Ser?Phe?Gln?His?Glu?Met?Ser?Gln?Glu?Gly?Phe?Ser?Ser?Ala?Leu?Thr

340 345 350

tcg?gat?gga?ccc?gtt?ctg?ggg?gcc?gyg?gga?agc?ttc?agc?tgg?tcc?gga 1104

Ser?Asp?Gly?Pro?Val?Leu?Gly?Ala?Xaa?Gly?Ser?Phe?Ser?Trp?Ser?Gly

355 360 365

ggt?gcc?ttc?tta?tat?ccc?cca?aat?acg?aga?ccc?acc?ttt?atc?aac?atg 1152

Gly?Ala?Phe?Leu?Tyr?Pro?Pro?Asn?Thr?Arg?Pro?Thr?Phe?Ile?Asn?Met

370 375 380

tct?cag?gag?aat?gtg?gac?atg?aga?gac?tcc?tac?ctg?ggt?tac?tcc?acc 1200

Ser?Gln?Glu?Asn?Val?Asp?Met?Arg?Asp?Ser?Tyr?Leu?Gly?Tyr?Ser?Thr

385 390 395 400

gca?gtg?gcc?ttt?tgg?aag?ggg?gtt?cac?agc?ctg?atc?ctg?ggg?gcc?ccg 1248

Ala?Val?Ala?Phe?Trp?Lys?Gly?Val?His?Ser?Leu?Ile?Leu?Gly?Ala?Pro

405 410 415

cgt?cac?cag?cac?acg?ggg?aag?gtt?gtc?atc?ttt?acc?cag?gaa?gcc?agg 1296

Arg?His?Gln?His?Thr?Gly?Lys?Val?Val?Ile?Phe?Thr?Gln?Glu?Ala?Arg

420 425 430

cat?tgg?agg?ccc?aag?tct?gaa?gtc?aga?ggg?aca?cag?atc?ggc?tcc?tac 1344

His?Trp?Arg?Pro?Lys?Ser?Glu?Val?Arg?Gly?Thr?Gln?Ile?Gly?Ser?Tyr

435 440 445

ttc?ggg?gcc?tct?ctc?tgt?tct?gtg?gac?gtg?gat?aga?gat?ggc?agc?acy 1392

Phe?Gly?Ala?Ser?Leu?Cys?Ser?Val?Asp?Val?Asp?Arg?Asp?Gly?Ser?Xaa

450 455 460

gac?ctg?gtc?ctg?atc?gga?gcc?ccc?cat?tac?tat?gag?cag?acc?cga?ggg 1440

Asp?Leu?Val?Leu?Ile?Gly?Ala?Pro?His?Tyr?Tyr?Glu?Gln?Thr?Arg?Gly

465 470 475 480

ggg?cag?gtc?tca?gtg?tkc?ccc?gtg?ccc?ggt?gtg?agg?ggc?agg?tgg?cag 1488

Gly?Gln?Val?Ser?Val?Xaa?Pro?Val?Pro?Gly?Val?Arg?Gly?Arg?Trp?Gln

485 490 495

tgt?gag?gcc?acc?ctc?cac?ggg?gag?cag?grc?cat?cct?tgg?ggc?cgc?ttt 1536

Cys?Glu?Ala?Thr?Leu?His?Gly?Glu?Gln?Xaa?His?Pro?Trp?Gly?Arg?Phe

500 505 510

ggg?gtg?gct?ctg?aca?gtg?ctg?ggg?gac?gta?aac?ggg?gac?aat?ctg?gca 1584

Gly?Val?Ala?Leu?Thr?Val?Leu?Gly?Asp?Val?Asn?Gly?Asp?Asn?Leu?Ala

515 520 525

gac?gtg?gct?att?ggt?gcc?cct?gga?gag?gag?gag?agc?aga?ggt?gct?gtc 1632

Asp?Val?Ala?Ile?Gly?Ala?Pro?Gly?Glu?Glu?Glu?Ser?Arg?Gly?Ala?Val

530 535 540

tac?ata?ttt?cat?gga?gcc?tcg?aga?ctg?gag?atc?atg?ccc?tca?ccc?agc 1680

Tyr?Ile?Phe?His?Gly?Ala?Ser?Arg?Leu?Glu?Ile?Met?Pro?Ser?Pro?Ser

545 550 555 560

cag?cgg?gtc?act?ggc?tcc?cag?ctc?tcc?ctg?aga?ctg?cag?tat?ttt?ggg 1728

Gln?Arg?Val?Thr?Gly?Ser?Gln?Leu?Ser?Leu?Arg?Leu?Gln?Tyr?Phe?Gly

565 570 575

cag?tca?ttg?agt?ggg?ggt?cag?gac?ctt?aca?cag?gat?ggc?ctg?gtg?gac 1776

Gln?Ser?Leu?Ser?Gly?Gly?Gln?Asp?Leu?Thr?Gln?Asp?Gly?Leu?Val?Asp

580 585 590

ctg?gcc?gtg?gga?gcc?cag?ggg?cac?gta?ctg?ctg?ctc?agg?agt?ctg?cct 1824

Leu?Ala?Val?Gly?Ala?Gln?Gly?His?Val?Leu?Leu?Leu?Arg?Ser?Leu?Pro

595 600 605

ctg?ctg?aaa?gtg?gag?ctc?tcc?ata?aga?ttc?gcc?ccc?atg?gag?gtg?gca 1872

Leu?Leu?Lys?Val?Glu?Leu?Ser?Ile?Arg?Phe?Ala?Pro?Met?Glu?Val?Ala

610 615 620

aag?gct?gtg?tac?cag?tgc?tgg?gaa?agg?act?ccc?act?gtc?ctc?gaa?gct 1920

Lys?Ala?Val?Tyr?Gln?Cys?Trp?Glu?Arg?Thr?Pro?Thr?Val?Leu?Glu?Ala

625 630 635 640

gga?gag?gcc?act?gtc?tgt?ctc?act?gtc?cac?aaa?ggc?tca?cct?gac?ctg 1968

Gly?Glu?Ala?Thr?Val?Cys?Leu?Thr?Val?His?Lys?Gly?Ser?Pro?Asp?Leu

645 650 655

tta?ggt?aat?gtc?caa?ggc?tct?gtc?agg?tat?gat?ctg?gcg?tta?gat?ccg 2016

Leu?Gly?Asn?Val?Gln?Gly?Ser?Val?Arg?Tyr?Asp?Leu?Ala?Leu?Asp?Pro

660 665 670

ggc?cgc?ctg?att?tct?cgt?gcc?att?ttt?gat?gag?act?aag?aac?tgc?act 2064

Gly?Arg?Leu?Ile?Ser?Arg?Ala?Ile?Phe?Asp?Glu?Thr?Lys?Asn?Cys?Thr

675 680 685

ttg?acg?gga?agg?aag?act?ctg?ggg?ctt?ggt?gat?cac?tgc?gaa?aca?gtg 2112

Leu?Thr?Gly?Arg?Lys?Thr?Leu?Gly?Leu?Gly?Asp?His?Cys?Glu?Thr?Val

690 695 700

aag?ctg?ctt?ttg?ccg?gac?tgt?gtg?gaa?gat?gca?gtg?agc?cct?atc?atc 2160

Lys?Leu?Leu?Leu?Pro?Asp?Cys?Val?Glu?Asp?Ala?Val?Ser?Pro?Ile?Ile

705 710 715 720

ctg?cgc?ctc?aac?ttt?tcc?ctg?gtg?aga?gac?tct?gct?tca?ccc?agg?aac 2208

Leu?Arg?Leu?Asn?Phe?Ser?Leu?Val?Arg?Asp?Ser?Ala?Ser?Pro?Arg?Asn

725 730 735

ctg?cat?cct?gtg?ctg?gct?gtg?ggc?tca?caa?gac?cac?ata?act?gct?tct 2256

Leu?His?Pro?Val?Leu?Ala?Val?Gly?Ser?Gln?Asp?His?Ile?Thr?Ala?Ser

740 745 750

ctg?ccg?ttt?gag?aag?aac?tgt?aag?caa?gaa?ctc?ctg?tgt?gag?ggg?gac 2304

Leu?Pro?Phe?Glu?Lys?Asn?Cys?Lys?Gln?Glu?Leu?Leu?Cys?Glu?Gly?Asp

755 760 765

ctg?ggc?atc?agc?ttt?aac?ttc?tca?ggc?ctg?cag?gtc?ttg?gtg?gtg?gga 2352

Leu?Gly?Ile?Ser?Phe?Asn?Phe?Ser?Gly?Leu?Gln?Val?Leu?Val?Val?Gly

770 775 780

ggc?tcc?cca?gag?ctc?act?gtg?aca?gtc?act?gtg?tgg?aat?gag?ggt?gag 2400

Gly?Ser?Pro?Glu?Leu?Thr?Val?Thr?Val?Thr?Val?Trp?Asn?Glu?Gly?Glu

785 790 795 800

gac?agc?tat?gga?act?tta?gtc?aag?ttc?tac?tac?cca?gca?ggg?cta?tct 2448

Asp?Ser?Tyr?Gly?Thr?Leu?Val?Lys?Phe?Tyr?Tyr?Pro?Ala?Gly?Leu?Ser

805 810 815

tac?cga?cgg?gta?aca?ggg?act?cag?gag?cct?cat?cag?tac?cca?cta?cgc 2496

Tyr?Arg?Arg?Val?Thr?Gly?Thr?Gln?Gln?Pro?His?Gln?Tyr?Pro?Leu?Arg

820 825 830

ttg?gcc?tgt?gag?gct?gag?ccc?gct?gcc?cag?gag?gac?ctg?agg?agc?agc 2544

Leu?Ala?Cys?Glu?Ala?Glu?Pro?Ala?Ala?Gln?Glu?Asp?Leu?Arg?Ser?Ser

835 840 845

agc?tgt?agc?att?aat?cac?ccc?atc?ttc?cga?gaa?ggt?gca?aag?acc?acc 2592

Ser?Cys?Ser?Ile?Asn?His?Pro?Ile?Phe?Arg?Glu?Gly?Ala?Lys?Thr?Thr

850 855 860

ttc?atg?atc?aca?ttc?gat?gtc?tcc?tac?aag?gcc?ttc?cta?gga?gac?agg 2640

Phe?Met?Ile?Thr?Phe?Asp?Val?Ser?Tyr?Lys?Ala?Phe?Leu?Gly?Asp?Arg

865 870 875 880

ttg?ctt?ctg?agg?gcc?aaa?gcc?agc?agt?gag?aat?aat?aag?cct?gat?acc 2688

Leu?Leu?Leu?Arg?Ala?Lys?Ala?Ser?Ser?Glu?Asn?Asn?Lys?Pro?Asp?Thr

885 890 895

aac?aag?act?gcc?ttc?cag?ctg?gag?ctc?cca?gtg?aag?tac?acc?gtc?tat 2736

Asn?Lys?Thr?Ala?Phe?Gln?Leu?Glu?Leu?Pro?Val?Lys?Tyr?Thr?Val?Tyr

900 905 910

acc?ctg?atc?agt?agg?caa?gaa?gat?tcc?acc?aac?cat?gtc?aac?ttt?tca 2784

Thr?Leu?Ile?Ser?Arg?Gln?Glu?Asp?Ser?Thr?Asn?His?Val?Asn?Phe?Ser

915 920 925

tct?tcc?cac?ggg?ggg?aga?agg?caa?gaa?gcc?gca?cat?cgc?tat?cgt?gtg 2832

Ser?Ser?His?Gly?Gly?Arg?Arg?Gln?Glu?Ala?Ala?His?Arg?Tyr?Arg?Val

930 935 940

aat?aac?ctg?agt?cca?ctg?aag?ctg?gcc?gtc?aga?gtt?aac?ttc?tgg?gtc 2880

Asn?Asn?Leu?Ser?Pro?Leu?Lys?Leu?Ala?Val?Arg?Val?Asn?Phe?Trp?Val

945 950 955 960

cct?gtc?ctt?ctg?aac?ggt?gtg?gct?gtg?tgg?gac?gtg?act?ctg?agc?agc 2928

Pro?Val?Leu?Leu?Asn?Gly?Val?Ala?Val?Trp?Asp?Val?Thr?Leu?Ser?Ser

965 970 975

cca?gca?cag?ggt?gtc?tcc?tgc?gtg?tcc?cag?atg?aaa?cct?cct?cag?aat 2976

Pro?Ala?Gln?Gly?Val?Ser?Cys?Val?Ser?Gln?Met?Lys?Pro?Pro?Gln?Asn

980 985 990

ccc?gac?ttt?ctg?acc?cag?att?cag?aga?cgt?tct?gtg?ctg?gac?tgc?tcc 3024

Pro?Asp?Phe?Leu?Thr?Gln?Ile?Gln?Arg?Arg?Ser?Val?Leu?Asp?Cys?Ser

995 1000 1005

att?gct?gac?tgc?ctg?cac?tcc?cgc?tgt?gac?atc?ccc?tcc?ttg?gac?atc 3072

Ile?Ala?Asp?Cys?Leu?His?Ser?Arg?Cys?Asp?Ile?Pro?Ser?Leu?Asp?Ile

1010 1015 1020

cag?gat?gaa?ctt?gac?ttc?att?ctg?agg?ggc?aac?ctc?agc?ttc?ggc?tgg 3120

Gln?Asp?Glu?Leu?Asp?Phe?Ile?Leu?Arg?Gly?Asn?Leu?Ser?Phe?Gly?Trp

1025 1030 1035 1040

gtc?agt?cag?aca?ttg?cag?gaa?aag?gtg?ttg?ctt?gtg?agt?gag?gct?gaa 3168

Val?Ser?Gln?Thr?Leu?Gln?Glu?Lys?Val?Leu?Leu?Val?Ser?Glu?Ala?Glu

1045 1050 1055

atc?act?ttc?gac?aca?tct?gtg?tac?tcc?cag?ctg?cca?gga?cag?gag?gca 3216

Ile?Thr?Phe?Asp?Thr?Ser?Val?Tyr?Ser?Gln?Leu?Pro?Gly?Gln?Glu?Ala

1060 1065 1070

ttt?ctg?aga?gcc?cag?gtg?gag?aca?acg?tta?gaa?gaa?tac?gtg?gtc?tat 3264

Phe?Leu?Arg?Ala?Gln?Val?Glu?Thr?Thr?Leu?Glu?Glu?Tyr?Val?Val?Tyr

1075 1080 1085

gag?ccc?atc?ttc?ctc?gtg?gcg?ggc?agc?tcg?gtg?gga?ggt?ctg?ctg?tta 3312

Glu?Pro?Ile?Phe?Leu?Val?Ala?Gly?Ser?Ser?Val?Gly?Gly?Leu?Leu?Leu

1090 1095 1100

ctg?gct?ctc?atc?aca?gtg?gta?ctg?tac?aag?ctt?ggc?tyc?tyc?aaa?cgt 3360

Leu?Ala?Leu?Ile?Thr?Val?Val?Leu?Tyr?Lys?Leu?Gly?Xaa?Xaa?Lys?Arg

1105 1110 1115 1120

cag?tac?aaa?gaa?atg?ctg?gac?ggc?aag?gct?gca?gat?cct?gtc?aca?gcc 3408

Gln?Tyr?Lys?Glu?Met?Leu?Asp?Gly?Lys?Ala?Ala?Asp?Pro?Val?Thr?Ala

1125 1130 1135

ggc?cag?gca?gat?ttc?ggc?tgt?gag?act?cct?cca?tat?ctc?gtg?agc 3453

Gly?Gln?Ala?Asp?Phe?Gly?Cys?Glu?Thr?Pro?Pro?Tyr?Leu?Val?Ser

1140 1145 1150

taggaatcca?ctctcctgcc?tatctctgna?atgaagattg?gtcctgccta?tgagtctact 3513

ggcatgggaa?cgagt 3528

<210>37

<211>1151

<212>PRT

＜213〉rat

<400>37

Gly?Trp?Ala?Leu?Ala?Ser?Cys?His?Gly?Ser?Asn?Leu?Asp?Val?Glu?Glu

1 5 10 15

Pro?Ile?Val?Phe?Arg?Glu?Asp?Ala?Ala?Ser?Phe?Gly?Gln?Thr?Val?Val

20 25 30

Gln?Phe?Gly?Gly?Ser?Arg?Leu?Val?Val?Gly?Ala?Pro?Leu?Glu?Ala?Val

35 40 45

Ala?Val?Asn?Gln?Thr?Gly?Arg?Leu?Tyr?Asp?Cys?Ala?Pro?Ala?Thr?Gly

50 55 60

Met?Cys?Gln?Pro?Ile?Val?Leu?Arg?Ser?Pro?Leu?Glu?Ala?Val?Asn?Met

65 70 75 80

Ser?Leu?Gly?Leu?Ser?Leu?Val?Thr?Ala?Thr?Asn?Asn?Ala?Gln?Leu?Leu

85 90 95

Ala?Cys?Gly?Pro?Thr?Ala?Gln?Arg?Ala?Cys?Val?Lys?Asn?Met?Tyr?Ala

100 105 110

Lys?Gly?Ser?Cys?Leu?Leu?Leu?Gly?Ser?Ser?Leu?Gln?Phe?Ile?Gln?Ala

115 120 125

Val?Pro?Ala?Ser?Met?Pro?Glu?Cys?Pro?Arg?Gln?Glu?Met?Asp?Ile?Ala

130 135 140

Phe?Leu?Ile?Asp?Gly?Ser?Gly?Ser?Ile?Asn?Gln?Arg?Asp?Phe?Ala?Gln

145 150 155 160

Met?Lys?Asp?Phe?Val?Lys?Ala?Leu?Met?Gly?Glu?Phe?Ala?Ser?Thr?Ser

165 170 175

Thr?Leu?Phe?Ser?Leu?Met?Gln?Tyr?Ser?Asn?Ile?Leu?Lys?Thr?His?Phe

180 185 190

Thr?Phe?Thr?Glu?Phe?Lys?Asn?Ile?Leu?Asp?Pro?Gln?Ser?Leu?Val?Asp

195 200 205

Pro?Ile?Val?Gln?Leu?Gln?Gly?Leu?Thr?Tyr?Thr?Ala?Thr?Gly?Ile?Arg

210 215 220

Thr?Val?Met?Glu?Glu?Leu?Phe?His?Ser?Lys?Asn?Gly?Ser?Arg?Lys?Ser

225 230 235 240

Ala?Lys?Lys?Ile?Leu?Leu?Val?Ile?Thr?Asp?Gly?Gln?Lys?Tyr?Arg?Asp

245 250 255

Pro?Leu?Glu?Tyr?Ser?Asp?Val?Ile?Pro?Ala?Ala?Asp?Lys?Ala?Gly?Ile

260 265 270

Ile?Arg?Tyr?Ala?Ile?Gly?Val?Gly?Asp?Ala?Phe?Gln?Glu?Pro?Thr?Ala

275 280 285

Leu?Lys?Glu?Leu?Asn?Thr?Ile?Gly?Ser?Ala?Pro?Pro?Gln?Asp?His?Val

290 295 300

Phe?Lys?Val?Gly?Asn?Phe?Ala?Ala?Leu?Arg?Ser?Ile?Gln?Arg?Gln?Leu

305 310 315 320

Gln?Glu?Lys?Ile?Phe?Ala?Ile?Glu?Gly?Thr?Gln?Ser?Arg?Ser?Ser?Ser

325 330 335

Ser?Phe?Gln?His?Glu?Met?Ser?Gln?Glu?Gly?Phe?Ser?Ser?Ala?Leu?Thr

340 345 350

Ser?Asp?Gly?Pro?Val?Leu?Gly?Ala?Xaa?Gly?Ser?Phe?Ser?Trp?Ser?Gly

355 360 365

Gly?Ala?Phe?Leu?Tyr?Pro?Pro?Asn?Thr?Arg?Pro?Thr?Phe?Ile?Asn?Met

370 375 380

Ser?Gln?Glu?Asn?Val?Asp?Met?Arg?Asp?Ser?Tyr?Leu?Gly?Tyr?Ser?Thr

385 390 395 400

Ala?Val?Ala?Phe?Trp?Lys?Gly?Val?His?Ser?Leu?Ile?Leu?Gly?Ala?Pro

405 410 415

Arg?His?Gln?His?Thr?Gly?Lys?Val?Val?Ile?Phe?Thr?Gln?Glu?Ala?Arg

420 425 430

His?Trp?Arg?Pro?Lys?Ser?Glu?Val?Arg?Gly?Thr?Gln?Ile?Gly?Ser?Tyr

435 440 445

Phe?Gly?Ala?Ser?Leu?Cys?Ser?Val?Asp?Val?Asp?Arg?Asp?Gly?Ser?Xaa

450 455 460

Asp?Leu?Val?Leu?Ile?Gly?Ala?Pro?His?Tyr?Tyr?Glu?Gln?Thr?Arg?Gly

465 470 475 480

Gly?Gln?Val?Ser?Val?Xaa?Pro?Val?Pro?Gly?Val?Arg?Gly?Arg?Trp?Gln

485 490 495

Cys?Glu?Ala?Thr?Leu?His?Gly?Glu?Gln?Xaa?His?Pro?Trp?Gly?Arg?Phe

500 505 510

Gly?Val?Ala?Leu?Thr?Val?Leu?Gly?Asp?Val?Asn?Gly?Asp?Asn?Leu?Ala

515 520 525

Asp?Val?Ala?Ile?Gly?Ala?Pro?Gly?Glu?Glu?Glu?Ser?Arg?Gly?Ala?Val

530 535 540

Tyr?Ile?Phe?His?Gly?Ala?Ser?Arg?Leu?Glu?Ile?Met?Pro?Ser?Pro?Ser

545 550 555 560

Gln?Arg?Val?Thr?Gly?Ser?Gln?Leu?Ser?Leu?Arg?Leu?Gln?Tyr?Phe?Gly

565 570 575

Gln?Ser?Leu?Ser?Gly?Gly?Gln?Asp?Leu?Thr?Gln?Asp?Gly?Leu?Val?Asp

580 585 590

Leu?Ala?Val?Gly?Ala?Gln?Gly?His?Val?Leu?Leu?Leu?Arg?Ser?Leu?Pro

595 600 605

Leu?Leu?Lys?Val?Glu?Leu?Ser?Ile?Arg?Phe?Ala?Pro?Met?Glu?Val?Ala

610 615 620

Lys?Ala?Val?Tyr?Gln?Cys?Trp?Glu?Arg?Thr?Pro?Thr?Val?Leu?Glu?Ala

625 630 635 640

Gly?Glu?Ala?Thr?Val?Cys?Leu?Thr?Val?His?Lys?Gly?Ser?Pro?Asp?Leu

645 650 655

Leu?Gly?Asn?Val?Gln?Gly?Ser?Val?Arg?Tyr?Asp?Leu?Ala?Leu?Asp?Pro

660 665 670

Gly?Arg?Leu?Ile?Ser?Arg?Ala?Ile?Phe?Asp?Glu?Thr?Lys?Asn?Cys?Thr

675 680 685

Leu?Thr?Gly?Arg?Lys?Thr?Leu?Gly?Leu?Gly?Asp?His?Cys?Glu?Thr?Val

690 695 700

Lys?Leu?Leu?Leu?Pro?Asp?Cys?Val?Glu?Asp?Ala?Val?Ser?Pro?Ile?Ile

705 710 715 720

Leu?Arg?Leu?Asn?Phe?Ser?Leu?Val?Arg?Asp?Ser?Ala?Ser?Pro?Arg?Asn

725 730 735

Leu?His?Pro?Val?Leu?Ala?Val?Gly?Ser?Gln?Asp?His?Ile?Thr?Ala?Ser

740 745 750

Leu?Pro?Phe?Glu?Lys?Asn?Cys?Lys?Gln?Glu?Leu?Leu?Cys?Glu?Gly?Asp

755 760 765

Leu?Gly?Ile?Ser?Phe?Asn?Phe?Ser?Gly?Leu?Gln?Val?Leu?Val?Val?Gly

770 775 780

Gly?Ser?Pro?Glu?Leu?Thr?Val?Thr?Val?Thr?Val?Trp?Asn?Glu?Gly?Glu

785 790 795 800

Asp?Ser?Tyr?Gly?Thr?Leu?Val?Lys?Phe?Tyr?Tyr?Pro?Ala?Gly?Leu?Ser

805 810 815

Tyr?Arg?Arg?Val?Thr?Gly?Thr?Gln?Gln?Pro?His?Gln?Tyr?Pro?Leu?Arg

820 825 830

Leu?Ala?Cys?Glu?Ala?Glu?Pro?Ala?Ala?Gln?Glu?Asp?Leu?Arg?Ser?Ser

835 840 845

Ser?Cys?Ser?Ile?Asn?His?Pro?Ile?Phe?Arg?Glu?Gly?Ala?Lys?Thr?Thr

850 855 860

Phe?Met?Ile?Thr?Phe?Asp?Val?Ser?Tyr?Lys?Ala?Phe?Leu?Gly?Asp?Arg

865 870 875 880

Leu?Leu?Leu?Arg?Ala?Lys?Ala?Ser?Ser?Glu?Asn?Asn?Lys?Pro?Asp?Thr

885 890 895

Asn?Lys?Thr?Ala?Phe?Gln?Leu?Glu?Leu?Pro?Val?Lys?Tyr?Thr?Val?Tyr

900 905 910

Thr?Leu?Ile?Ser?Arg?Gln?Glu?Asp?Ser?Thr?Asn?His?Val?Asn?Phe?Ser

915 920 925

Ser?Ser?His?Gly?Gly?Arg?Arg?Gln?Glu?Ala?Ala?His?Arg?Tyr?Arg?Val

930 935 940

Asn?Asn?Leu?Ser?Pro?Leu?Lys?Leu?Ala?Val?Arg?Val?Asn?Phe?Trp?Val

945 950 955 960

Pro?Val?Leu?Leu?Asn?Gly?Val?Ala?Val?Trp?Asp?Val?Thr?Leu?Ser?Ser

965 970 975

Pro?Ala?Gln?Gly?Val?Ser?Cys?Val?Ser?Gln?Met?Lys?Pro?Pro?Gln?Asn

980 985 990

Pro?Asp?Phe?Leu?Thr?Gln?Ile?Gln?Arg?Arg?Ser?Val?Leu?Asp?Cys?Ser

995 1000 1005

Ile?Ala?Asp?Cys?Leu?His?Ser?Arg?Cys?Asp?Ile?Pro?Ser?Leu?Asp?Ile

1010 1015 1020

Gln?Asp?Glu?Leu?Asp?Phe?Ile?Leu?Arg?Gly?Asn?Leu?Ser?Phe?Gly?Trp

1025 1030 1035 1040

Val?Ser?Gln?Thr?Leu?Gln?Glu?Lys?Val?Leu?Leu?Val?Ser?Glu?Ala?Glu

1045 1050 1055

Ile?Thr?Phe?Asp?Thr?Ser?Val?Tyr?Ser?Gln?Leu?Pro?Gly?Gln?Glu?Ala

1060 1065 1070

Phe?Leu?Arg?Ala?Gln?Val?Glu?Thr?Thr?Leu?Glu?Glu?Tyr?Val?Val?Tyr

1075 1080 1085

Glu?Pro?Ile?Phe?Leu?Val?Ala?Gly?Ser?Ser?Val?Gly?Gly?Leu?Leu?Leu

1090 1095 1100

Leu?Ala?Leu?Ile?Thr?Val?Val?Leu?Tyr?Lys?Leu?Gly?Xaa?Xaa?Lys?Arg

1105 1110 1115 1120

Gln?Tyr?Lys?Glu?Met?Leu?Asp?Gly?Lys?Ala?Ala?Asp?Pro?Val?Thr?Ala

1125 1130 1135

Gly?Gln?Ala?Asp?Phe?Gly?Cys?Glu?Thr?Pro?Pro?Tyr?Leu?Val?Ser

1140 1145 1150

<210>38

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>38

gtccaagctg?tcatgggcca?g 21

<210>39

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>39

gtccagcaga?ctgaagagca?cgg 23

<210>40

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>40

tgtaaaacga?cggccagt 18

<210>41

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>41

ggaaacagct?atgaccatg 19

<210>42

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>42

ggacatgttc?actgcctcta?gg 22

<210>43

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>43

ggcggacagt?cagacgactg?tcctg 25

<210>44

<211>38

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>44

ctggttcggc?ccacctctga?aggttccaga?atcgatag 38

<210>45

<211>3519

<212>DNA

＜213〉mice

<220>

<221>CDS

<222>(52)..(3516)

<220>

＜223〉artificial sequence note: primer

<400>45

gctttctgaa?ggttccagaa?tcgatagtga?attcgtgggc?actgctcaga?t?atg?gtc 57

Met?Val

1

cgt?gga?gtt?gtg?atc?ctc?ctg?tgt?ggc?tgg?gcc?ctg?gct?tcc?tgt?cat 105

Arg?Gly?Val?Val?Ile?Leu?Leu?Cys?Gly?Trp?Ala?Leu?Ala?Ser?Cys?His

5 10 15

ggg?tct?aac?ctg?gat?gtg?gag?aag?ccc?gtc?gtg?ttc?aaa?gag?gat?gca 153

Gly?Ser?Asn?Leu?Asp?Val?Glu?Lys?Pro?Val?Val?Phe?Lys?Glu?Asp?Ala

20 25 30

gcc?agc?ttc?gga?cag?act?gtg?gtg?cag?ttt?ggt?gga?tct?cga?ctc?gtg 201

Ala?Ser?Phe?Gly?Gln?Thr?Val?Val?Gln?Phe?Gly?Gly?Ser?Arg?Leu?Val

35 40 45 50

gtg?gga?gcc?cct?ctg?gag?gcg?gtg?gca?gtc?aac?caa?aca?gga?cag?tcg 249

Val?Gly?Ala?Pro?Leu?Glu?Ala?Val?Ala?Val?Asn?Gln?Thr?Gly?Gln?Ser

55 60 65

tct?gac?tgt?ccg?cct?gcc?act?ggc?gtg?tgc?cag?ccc?atc?tta?ctg?cac 297

Ser?Asp?Cys?Pro?Pro?Ala?Thr?Gly?Val?Cys?Gln?Pro?Ile?Leu?Leu?His

70 75 80

att?ccc?cta?gag?gca?gtg?aac?atg?tcc?ctg?ggc?ctg?tct?ctg?gtg?gct 345

Ile?Pro?Leu?Glu?Ala?Val?Asn?Met?Ser?Leu?Gly?Leu?Ser?Leu?Val?Ala

85 90 95

gac?acc?aat?aac?tcc?cag?ttg?ctg?gct?tgt?ggt?cca?act?gca?cag?aga 393

Asp?Thr?Asn?Asn?Ser?Gln?Leu?Leu?Ala?Cys?Gly?Pro?Thr?Ala?Gln?Arg

100 105 110

gct?tgt?gca?aag?aac?atg?tat?gca?aaa?ggt?tcc?tgc?ctc?ctt?ctg?ggc 441

Ala?Cys?Ala?Lys?Asn?Met?Tyr?Ala?Lys?Gly?Ser?Cys?Leu?Leu?Leu?Gly

115 120 125 130

tcc?agc?ttg?cag?ttc?atc?cag?gca?atc?cct?gct?acc?atg?cca?gag?tgt 489

Ser?Ser?Leu?Gln?Phe?Ile?Gln?Ala?Ile?Pro?Ala?Thr?Met?Pro?Glu?Cys

135 140 145

cca?gga?caa?gag?atg?gac?att?gct?ttc?ctg?att?gat?ggc?tcc?ggc?agc 537

Pro?Gly?Gln?Glu?Met?Asp?Ile?Ala?Phe?Leu?Ile?Asp?Gly?Ser?Gly?Ser

150 155 160

att?gat?caa?agt?gac?ttt?acc?cag?atg?aag?gac?ttc?gtc?aaa?gct?ttg 585

Ile?Asp?Gln?Ser?Asp?Phe?Thr?Gln?Met?Lys?Asp?Phe?Val?Lys?Ala?Leu

165 170 175

atg?ggc?cag?ttg?gcg?agc?acc?agc?acc?tcg?ttc?tcc?ctg?atg?caa?tac 633

Met?Gly?Gln?Leu?Ala?Ser?Thr?Ser?Thr?Ser?Phe?Ser?Leu?Met?Gln?Tyr

180 185 190

tca?aac?atc?ctg?aag?act?cat?ttt?acc?ttc?acg?gaa?ttc?aag?agc?agc 681

Ser?Asn?Ile?Leu?Lys?Thr?His?Phe?Thr?Phe?Thr?Glu?Phe?Lys?Ser?Ser

195 200 205 210

ctg?agc?cct?cag?agc?ctg?gtg?gat?gcc?atc?gtc?cag?ctc?caa?ggc?ctg 729

Leu?Ser?Pro?Gln?Ser?Leu?Val?Asp?Ala?Ile?Val?Gln?Leu?Gln?Gly?Leu

215 220 225

acg?tac?aca?gcc?tcg?ggc?atc?cag?aaa?gtg?gtg?aaa?gag?cta?ttt?cat 777

Thr?Tyr?Thr?Ala?Ser?Gly?Ile?Gln?Lys?Val?Val?Lys?Glu?Leu?Phe?His

230 235 240

agc?aag?aat?ggg?gcc?cga?aaa?agt?gcc?aag?aag?ata?cta?att?gtc?atc 825

Ser?Lys?Asn?Gly?Ala?Arg?Lys?Ser?Ala?Lys?Lys?Ile?Leu?Ile?Val?Ile

245 250 255

aca?gat?ggg?cag?aaa?ttc?aga?gac?ccc?ctg?gag?tat?aga?cat?gtc?atc 873

Thr?Asp?Gly?Gln?Lys?Phe?Arg?Asp?Pro?Leu?Glu?Tyr?Arg?His?Val?Ile

260 265 270

cct?gaa?gca?gag?aaa?gct?ggg?atc?att?cgc?tat?gct?ata?ggg?gtg?gga 921

Pro?Glu?Ala?Glu?Lys?Ala?Gly?Ile?Ile?Arg?Tyr?Ala?Ile?Gly?Val?Gly

275 280 285 290

gat?gcc?ttc?cgg?gaa?ccc?act?gcc?cta?cag?gag?ctg?aac?acc?att?ggc 969

Asp?Ala?Phe?Arg?Glu?Pro?Thr?Ala?Leu?Gln?Glu?Leu?Asn?Thr?Ile?Gly

295 300 305

tca?gct?ccc?tcg?cag?gac?cac?gtg?ttc?aag?gtg?ggc?aat?ttt?gta?gca 1017

Ser?Ala?Pro?Ser?Gln?Asp?His?Val?Phe?Lys?Val?Gly?Asn?Phe?Val?Ala

310 315 320

ctt?cgc?agc?atc?cag?cgg?caa?att?cag?gag?aaa?atc?ttt?gcc?att?gaa 1065

Leu?Arg?Ser?Ile?Gln?Arg?Gln?Ile?Gln?Glu?Lys?Ile?Phe?Ala?Ile?Glu

325 330 335

gga?acc?gaa?tca?agg?tca?agt?agt?tcc?ttt?cag?cac?gag?atg?tca?caa 1113

Gly?Thr?Glu?Ser?Arg?Ser?Ser?Ser?Ser?Phe?Gln?His?Glu?Met?Ser?Gln

340 345 350

gaa?ggt?ttc?agc?tca?gct?ctc?tca?atg?gat?gga?cca?gtt?ctg?ggg?gct 1161

Glu?Gly?Phe?Ser?Ser?Ala?Leu?Ser?Met?Asp?Gly?Pro?Val?Leu?Gly?Ala

355 360 365 370

gtg?gga?ggc?ttc?agc?tgg?tct?gga?ggt?gcc?ttc?ttg?tac?ccc?tca?aat 1209

Val?Gly?Gly?Phe?Ser?Trp?Ser?Gly?Gly?Ala?Phe?Leu?Tyr?Pro?Ser?Asn

375 380 385

atg?aga?tcc?acc?ttc?atc?aac?atg?tct?cag?gag?aac?gag?gat?atg?agg 1257

Met?Arg?Ser?Thr?Phe?Ile?Asn?Met?Ser?Gln?Glu?Asn?Glu?Asp?Met?Arg

390 395 400

gac?gct?tac?ctg?ggt?tac?tcc?acc?gca?ctg?gcc?ttt?tgg?aag?ggg?gtc 1305

Asp?Ala?Tyr?Leu?Gly?Tyr?Ser?Thr?Ala?Leu?Ala?Phe?Trp?Lys?Gly?Val

405 410 415

cac?agc?ctg?atc?ctg?ggg?gcc?cct?cgc?cac?cag?cac?acg?ggg?aag?gtt 1353

His?Ser?Leu?Ile?Leu?Gly?Ala?Pro?Arg?His?Gln?His?Thr?Gly?Lys?Val

420 425 430

gtc?atc?ttt?acc?cag?gaa?tcc?agg?cac?tgg?agg?ccc?aag?tct?gaa?gtc 1401

Val?Ile?Phe?Thr?Gln?Glu?Ser?Arg?His?Trp?Arg?Pro?Lys?Ser?Glu?Val

435 440 445 450

aga?ggg?aca?cag?atc?ggc?tcc?tac?ttt?ggg?gca?tct?ctc?tgt?tct?gtg 1449

Arg?Gly?Thr?Gln?Ile?Gly?Ser?Tyr?Phe?Gly?Ala?Ser?Leu?Cys?Ser?Val

455 460 465

gac?atg?gat?aga?gat?ggc?agc?act?gac?ctg?gtc?ctg?att?gga?gtc?ccc 1497

Asp?Met?Asp?Arg?Asp?Gly?Ser?Thr?Asp?Leu?Val?Leu?Ile?Gly?Val?Pro

470 475 480

cat?tac?tat?gag?cac?acc?cga?ggg?ggg?cag?gtg?tcg?gtg?tgc?ccc?atg 1545

His?Tyr?Tyr?Glu?His?Thr?Arg?Gly?Gly?Gln?Val?Ser?Val?Cys?Pro?Met

485 490 495

cct?ggt?gtg?agg?agc?agg?tgg?cat?tgt?ggg?acc?acc?ctc?cat?ggg?gag 1593

Pro?Gly?Val?Arg?Ser?Arg?Trp?His?Cys?Gly?Thr?Thr?Leu?His?Gly?Glu

500 505 510

cag?ggc?cat?cct?tgg?ggc?cgc?ttt?ggg?gcg?gct?ctg?aca?gtg?cta?ggg 1641

Gln?Gly?His?Pro?Trp?Gly?Arg?Phe?Gly?Ala?Ala?Leu?Thr?Val?Leu?Gly

515 520 525 530

gac?gtg?aat?ggg?gac?agt?ctg?gcg?gat?gtg?gct?att?ggt?gca?ccc?gga 1689

Asp?Val?Asn?Gly?Asp?Ser?Leu?Ala?Asp?Val?Ala?Ile?Gly?Ala?Pro?Gly

535 540 545

gag?gag?gag?aac?aga?ggt?gct?gtc?tac?ata?ttt?cat?gga?gcc?tcg?aga 1737

Glu?Glu?Glu?Asn?Arg?Gly?Ala?Val?Tyr?Ile?Phe?His?Gly?Ala?Ser?Arg

550 555 560

cag?gac?atc?gct?ccc?tcg?cct?agc?cag?cgg?gtc?act?ggc?tcc?cag?ctc 1785

Gln?Asp?Ile?Ala?Pro?Ser?Pro?Ser?Gln?Arg?Val?Thr?Gly?Ser?Gln?Leu

565 570 575

ttc?ctg?agg?ctc?caa?tat?ttt?ggg?cag?tca?tta?agt?ggg?ggt?cag?gac 1833

Phe?Leu?Arg?Leu?Gln?Tyr?Phe?Gly?Gln?Ser?Leu?Ser?Gly?Gly?Gln?Asp

580 585 590

ctt?aca?cag?gat?ggc?ctg?gtg?gac?ctg?gcc?gtg?gga?gcc?cag?ggg?cac 1881

Leu?Thr?Gln?Asp?Gly?Leu?Val?Asp?Leu?Ala?Val?Gly?Ala?Gln?Gly?His

595 600 605 610

gtg?ctg?ctg?ctt?agg?agt?ctg?cct?ttg?ctg?aaa?gtg?ggg?atc?tcc?att 1929

Val?Leu?Leu?Leu?Arg?Ser?leu?Pro?Leu?Leu?Lys?Val?Gly?Ile?Ser?Ile

615 620 625

aga?ttt?gcc?ccc?tca?gag?gtg?gca?aag?act?gtg?tac?cag?tgc?tgg?gga 1977

Arg?Phe?Ala?Pro?Ser?Glu?Val?Ala?Lys?Thr?Val?Tyr?Gln?Cys?Trp?Gly

630 635 640

agg?act?ccc?act?gtc?ctc?gaa?gct?gga?gag?gcc?acc?gtc?tgt?ctc?act 2025

Arg?Thr?Pro?Thr?Val?Leu?Glu?Ala?Gly?Glu?Ala?Thr?Val?Cys?Leu?Thr

645 650 655

gtc?cgc?aaa?ggt?tca?cct?gac?ctg?tta?ggt?gat?gtc?caa?agc?tct?gtc 2073

Val?Arg?Lys?Gly?Ser?Pro?Asp?Leu?Leu?Gly?Asp?Val?Gln?Ser?Ser?Val

660 665 670

agg?tat?gat?ctg?gcg?ttg?gat?ccg?ggc?cgt?ctg?att?tct?cgt?gcc?att 2121

Arg?Tyr?Asp?Leu?Ala?Leu?Asp?Pro?Gly?Arg?Leu?Ile?Ser?Arg?Ala?Ile

675 680 685 690

ttt?gat?gag?acg?aag?aac?tgc?act?ttg?acc?cga?agg?aag?act?ctg?ggg 2169

Phe?Asp?Glu?Thr?Lys?Asn?Cys?Thr?Leu?Thr?Arg?Arg?Lys?Thr?Leu?Gly

695 700 705

ctt?ggt?gat?cac?tgc?gaa?aca?atg?aag?ctg?ctt?ttg?cca?gac?tgt?gtg 2217

Leu?Gly?Asp?His?Cys?Glu?Thr?Met?Lys?Leu?Leu?Leu?Pro?Asp?Cys?Val

710 715 720

gag?gat?gca?gtg?acc?cct?atc?atc?ctg?cgc?ctt?aac?tta?tcc?ctg?gca 2265

Glu?Asp?Ala?Val?Thr?Pro?Ile?Ile?Leu?Arg?Leu?Asn?Leu?Ser?Leu?Ala

725 730 735

ggg?gac?tct?gct?cca?tcc?agg?aac?ctt?cgt?cct?gtg?ctg?gct?gtg?ggc 2313

Gly?Asp?Ser?Ala?Pro?Ser?Arg?Asn?Leu?Arg?Pro?Val?Leu?Ala?Val?Gly

740 745 750

tca?caa?gac?cat?gta?aca?gct?tct?ttc?ccg?ttt?gag?aag?aac?tgt?gag 236l

Ser?Gln?Asp?His?Val?Thr?Ala?Ser?Phe?Pro?Phe?Glu?Lys?Asn?Cys?Glu

755 760 765 770

ggg?aac?ctg?ggc?gtc?agc?ttc?aac?ttc?tca?ggc?ctg?cag?gtc?ttg?gag 2409

Gly?Asn?Leu?Gly?Val?Ser?Phe?Asn?Phe?Ser?Gly?Leu?Gln?Val?Leu?Glu

775 780 785

gta?gga?agc?tcc?cca?gag?ctc?act?gtg?aca?gta?aca?gtt?tgg?aat?gag 2457

Val?Gly?Ser?Ser?Pro?Glu?Leu?Thr?Val?Thr?Val?Thr?Val?Trp?Asn?Glu

790 795 800

ggt?gag?gac?agc?tat?gga?acc?tta?atc?aag?ttc?tac?tac?cca?gca?gag 2505

Gly?Glu?Asp?Ser?Tyr?Gly?Thr?Leu?Ile?Lys?Phe?Tyr?Tyr?Pro?Ala?Glu

805 810 815

cta?tct?tac?cga?cgg?gtg?aca?aga?gcc?cag?caa?cct?cat?ccg?tac?cca 2553

Leu?Ser?Tyr?Arg?Arg?Val?Thr?Arg?Ala?Gln?Gln?Pro?His?Pro?Tyr?Pro

820 825 830

cta?cgc?ctg?gca?tgt?gag?gct?gag?ccc?acg?ggc?cag?gag?agc?ctg?agg 2601

Leu?Arg?Leu?Ala?Cys?Glu?Ala?Glu?Pro?Thr?Gly?Gln?Glu?Ser?Leu?Arg

835 840 845 850

agc?agc?agc?tgt?agc?atc?aat?cac?ccc?atc?ttc?cga?gaa?ggt?gcc?aag 2649

Ser?Ser?Ser?Cys?Ser?Ile?Asn?His?Pro?Ile?Phe?Arg?Glu?Gly?Ala?Lys

855 860 865

gcc?acc?ttc?atg?atc?aca?ttt?gat?gtc?tcc?tac?aag?gcc?ttc?ctg?gga 2697

Ala?Thr?Phe?Met?Ile?Thr?Phe?Asp?Val?Ser?Tyr?Lys?Ala?Phe?Leu?Gly

870 875 880

gac?agg?ttg?ctt?ctg?agg?gcc?agc?gca?agc?agt?gag?aat?aat?aag?cct 2745

Asp?Arg?Leu?Leu?Leu?Arg?Ala?Ser?Ala?Ser?Ser?Glu?Asn?Asn?Lys?Pro

885 890 895

gaa?acc?agc?aag?act?gcc?ttc?cag?ctg?gag?ctt?ccg?gtg?aag?tac?acg 2793

Glu?Thr?Ser?Lys?Thr?Ala?Phe?Gln?Leu?Glu?Leu?Pro?Val?Lys?Tyr?Thr

900 905 910

gtc?tat?acc?gtg?atc?agt?agg?cag?gaa?gat?tct?acc?aag?cat?ttc?aac 2841

Val?Tyr?Thr?Val?Ile?Ser?Arg?Gln?Glu?Asp?Ser?Thr?Lys?His?Phe?Asn

915 920 925 930

ttc?tca?tct?tcc?cac?ggg?gag?aga?cag?aaa?gag?gcc?gaa?cat?cga?tat 2889

Phe?Ser?Ser?Ser?His?Gly?Glu?Arg?Gln?Lys?Glu?Ala?Glu?His?Arg?Tyr

935 940 945

cgt?gtg?aat?aac?ctg?agt?cca?ttg?acg?ctg?gcc?atc?agc?gtt?aac?ttc 2937

Arg?Val?Asn?Asn?Leu?Ser?Pro?Leu?Thr?Leu?Ala?Ile?Ser?Val?Asn?Phe

950 955 960

tgg?gtc?ccc?atc?ctt?ctg?aat?ggt?gtg?gcc?gtg?tgg?gat?gtg?act?ctg 2985

Trp?Val?Pro?Ile?Leu?Leu?Asn?Gly?Val?Ala?Val?Trp?Asp?Val?Thr?Leu

965 970 975

agg?agc?cca?gca?cag?ggt?gtc?tcc?tgt?gtg?tca?cag?agg?gaa?cct?cct 3033

Arg?Ser?Pro?Ala?Gln?Gly?Val?Ser?Cys?Val?Ser?Gln?Arg?Glu?Pro?Pro

980 985 990

caa?cat?tcc?gac?ctt?ctg?acc?cag?atc?caa?gga?cgc?tct?gtg?ctg?gac 3081

Gln?His?Ser?Asp?Leu?Leu?Thr?Gln?Ile?Gln?Gly?Arg?Ser?Val?Leu?Asp

995 1000 1005 1010

tgc?gcc?atc?gcc?gac?tgc?ctg?cac?ctc?cgc?tgt?gac?atc?ccc?tcc?ttg 3129

Cys?Ala?Ile?Ala?Asp?Cys?Leu?His?Leu?Arg?Cys?Asp?Ile?Pro?Ser?Leu

1015 1020 1025

ggc?acc?ctg?gat?gag?ctt?gac?ttc?att?ctg?aag?ggc?aac?ctc?agc?ttc 3177

Gly?Thr?Leu?Asp?Glu?Leu?Asp?Phe?Ile?Leu?Lys?Gly?Asn?Leu?Ser?Phe

1030 1035 1040

ggc?tgg?atc?agt?cag?aca?ttg?cag?aaa?aag?gtg?ttg?ctc?ctg?agt?gag 3225

Gly?Trp?Ile?Ser?Gln?Thr?Leu?Gln?Lys?Lys?Val?Leu?Leu?Leu?Ser?Glu

1045 1050 1055

gct?gaa?atc?aca?ttc?aac?aca?tct?gtg?tat?tcc?cag?ctg?ccg?gga?cag 3273

Ala?Glu?Ile?Thr?Phe?Asn?Thr?Ser?Val?Tyr?Ser?Gln?Leu?Pro?Gly?Gln

1060 1065 1070

gag?gca?ttt?ctg?aga?gcc?cag?gtg?tca?acg?atg?cta?gaa?gaa?tac?gtg 3321

Glu?Ala?Phe?Leu?Arg?Ala?Gln?Val?Ser?Thr?Met?Leu?Glu?Glu?Tyr?Val

1075 1080 1085 1090

gtc?tat?gag?ccc?gtc?ttc?ctc?atg?gtg?ttc?agc?tca?gtg?gga?ggt?ctg 3369

Val?Tyr?Glu?Pro?Val?Phe?Leu?Met?Val?Phe?Ser?Ser?Val?Gly?Gly?Leu

1095 1100 1105

ctg?tta?ctg?gct?ctc?atc?act?gtg?gcg?ctg?tac?aag?ctt?ggc?ttc?ttc 3417

Leu?Leu?Leu?Ala?Leu?Ile?Thr?Val?Ala?Leu?Tyr?Lys?Leu?Gly?Phe?Phe

1110 1115 1120

aaa?cgt?cag?tat?aaa?gag?atg?ctg?gat?cta?cca?tct?gca?gat?cct?gac 3465

Lys?Arg?Gln?Tyr?Lys?Glu?Met?Leu?Asp?Leu?Pro?Ser?Ala?Asp?Pro?Asp

1125 1130 1135

cca?gcc?ggc?cag?gca?gat?tcc?aac?cat?gag?act?cct?cca?cat?ctc?acg 3513

Pro?Ala?Gly?Gln?Ala?Asp?Ser?Asn?His?Glu?Thr?Pro?Pro?His?Leu?Thr

1140 1145 1150

tcc?tag 3519

Ser

1155

<210>46

<211>1155

<212>PRT

＜213〉mice

<400>46

Met?Val?Arg?Gly?Val?Val?Ile?Leu?Leu?Cys?Gly?Trp?Ala?Leu?Ala?Ser

1 5 10 15

Cys?His?Gly?Ser?Asn?Leu?Asp?Val?Glu?Lys?Pro?Val?Val?Phe?Lys?Glu

20 25 30

Asp?Ala?Ala?Ser?Phe?Gly?Gln?Thr?Val?Val?Gln?Phe?Gly?Gly?Ser?Arg

35 40 45

Leu?Val?Val?Gly?Ala?Pro?Leu?Glu?Ala?Val?Ala?Val?Asn?Gln?Thr?Gly

50 55 60

Gln?Ser?Ser?Asp?Cys?Pro?Pro?Ala?Thr?Gly?Val?Cys?Gln?Pro?Ile?Leu

65 70 75 80

Leu?His?Ile?Pro?Leu?Glu?Ala?Val?Asn?Met?Ser?Leu?Gly?Leu?Ser?Leu

85 90 95

Val?Ala?Asp?Thr?Asn?Asn?Ser?Gln?Leu?Leu?Ala?Cys?Gly?Pro?Thr?Ala

100 105 110

Gln?Arg?Ala?Cys?Ala?Lys?Asn?Met?Tyr?Ala?Lys?Gly?Ser?Cys?Leu?Leu

115 120 125

Leu?Gly?Ser?Ser?Leu?Gln?Phe?Ile?Gln?Ala?Ile?Pro?Ala?Thr?Met?Pro

130 135 140

Glu?Cys?Pro?Gly?Gln?Glu?Met?Asp?Ile?Ala?Phe?Leu?Ile?Asp?Gly?Ser

145 150 155 160

Gly?Ser?Ile?Asp?Gln?Ser?Asp?Phe?Thr?Gln?Met?Lys?Asp?Phe?Val?Lys

165 170 175

Ala?Leu?Met?Gly?Gln?Leu?Ala?Ser?Thr?Ser?Thr?Ser?Phe?Ser?Leu?Met

180 185 190

Gln?Tyr?Ser?Asn?Ile?Leu?Lys?Thr?His?Phe?Thr?Phe?Thr?Glu?Phe?Lys

195 200 205

Ser?Ser?Leu?Ser?Pro?Gln?Ser?Leu?Val?Asp?Ala?Ile?Val?Gln?Leu?Gln

210 215 220

Gly?Leu?Thr?Tyr?Thr?Ala?Ser?Gly?Ile?Gln?Lys?Val?Val?Lys?Glu?Leu

225 230 235 240

Phe?His?Ser?Lys?Asn?Gly?Ala?Arg?Lys?Ser?Ala?Lys?Lys?Ile?Leu?Ile

245 250 255

Val?Ile?Thr?Asp?Gly?Gln?Lys?Phe?Arg?Asp?Pro?Leu?Glu?Tyr?Arg?His

260 265 270

Val?Ile?Pro?Glu?Ala?Glu?Lys?Ala?Gly?Ile?Ile?Arg?Tyr?Ala?Ile?Gly

275 280 285

Val?Gly?Asp?Ala?Phe?Arg?Glu?Pro?Thr?Ala?Leu?Gln?Glu?Leu?Asn?Thr

290 295 300

Ile?Gly?Ser?Ala?Pro?Ser?Gln?Asp?His?Val?Phe?Lys?Val?Gly?Asn?Phe

305 310 315 320

Val?Ala?Leu?Arg?Ser?Ile?Gln?Arg?Gln?Ile?Gln?Glu?Lys?Ile?Phe?Ala

325 330 335

Ile?Glu?Gly?Thr?Glu?Ser?Arg?Ser?Ser?Ser?Ser?Phe?Gln?His?Glu?Met

340 345 350

Ser?Gln?Glu?Gly?Phe?Ser?Ser?Ala?Leu?Ser?Met?Asp?Gly?Pro?Val?Leu

355 360 365

Gly?Ala?Val?Gly?Gly?Phe?Ser?Trp?Ser?Gly?Gly?Ala?Phe?Leu?Tyr?Pro

370 375 380

Ser?Asn?Met?Arg?Ser?Thr?Phe?Ile?Asn?Met?Ser?Gln?Glu?Asn?Glu?Asp

385 390 395 400

Met?Arg?Asp?Ala?Tyr?Leu?Gly?Tyr?Ser?Thr?Ala?Leu?Ala?Phe?Trp?Lys

405 410 415

Gly?Val?His?Ser?Leu?Ile?Leu?Gly?Ala?Pro?Arg?His?Gln?His?Thr?Gly

420 425 430

Lys?Val?Val?Ile?Phe?Thr?Gln?Glu?Ser?Arg?His?Trp?Arg?Pro?Lys?Ser

435 440 445

Glu?Val?Arg?Gly?Thr?Gln?Ile?Gly?Ser?Tyr?Phe?Gly?Ala?Ser?Leu?Cys

450 455 460

Ser?Val?Asp?Met?Asp?Arg?Asp?Gly?Ser?Thr?Asp?Leu?Val?Leu?Ile?Gly

465 470 475 480

Val?Pro?His?Tyr?Tyr?Glu?His?Thr?Arg?Gly?Gly?Gln?Val?Ser?Val?Cys

485 490 495

Pro?Met?Pro?Gly?Val?Arg?Ser?Arg?Trp?His?Cys?Gly?Thr?Thr?Leu?His

500 505 510

Gly?Glu?Gln?Gly?His?Pro?Trp?Gly?Arg?Phe?Gly?Ala?Ala?Leu?Thr?Val

515 520 525

Leu?Gly?Asp?Val?Asn?Gly?Asp?Ser?Leu?Ala?Asp?Val?Ala?Ile?Gly?Ala

530 535 540

Pro?Gly?Glu?Glu?Glu?Asn?Arg?Gly?Ala?Val?Tyr?Ile?Phe?His?Gly?Ala

545 550 555 560

Ser?Arg?Gln?Asp?Ile?Ala?Pro?Ser?Pro?Ser?Gln?Arg?Val?Thr?Gly?Ser

565 570 575

Gln?Leu?Phe?Leu?Arg?Leu?Gln?Tyr?Phe?Gly?Gln?Ser?Leu?Ser?Gly?Gly

580 585 590

Gln?Asp?Leu?Thr?Gln?Asp?Gly?Leu?Val?Asp?Leu?Ala?Val?Gly?Ala?Gln

595 600 605

Gly?His?Val?Leu?Leu?Leu?Arg?Ser?Leu?Pro?Leu?Leu?Lys?Val?Gly?Ile

610 615 620

Ser?Ile?Arg?Phe?Ala?Pro?Ser?Glu?Val?Ala?Lys?Thr?Val?Tyr?Gln?Cys

625 630 635 640

Trp?Gly?Arg?Thr?Pro?Thr?Val?Leu?Glu?Ala?Gly?Glu?Ala?Thr?Val?Cys

645 650 655

Leu?Thr?Val?Arg?Lys?Gly?Ser?Pro?Asp?Leu?Leu?Gly?Asp?Val?Gln?Ser

660 665 670

Ser?Val?Arg?Tyr?Asp?Leu?Ala?Leu?Asp?Pro?Gly?Arg?Leu?Ile?Ser?Arg

675 680 685

Ala?Ile?Phe?Asp?Glu?Thr?Lys?Asn?Cys?Thr?Leu?Thr?Arg?Arg?Lys?Thr

690 695 700

Leu?Gly?Leu?Gly?Asp?His?Cys?Glu?Thr?Met?Lys?Leu?Leu?Leu?Pro?Asp

705 710 715 720

Cys?Val?Glu?Asp?Ala?Val?Thr?Pro?Ile?Ile?Leu?Arg?Leu?Asn?Leu?Ser

725 730 735

Leu?Ala?Gly?Asp?Ser?Ala?Pro?Ser?Arg?Asn?Leu?Arg?Pro?Val?Leu?Ala

740 745 750

Val?Gly?Ser?Gln?Asp?His?Val?Thr?Ala?Ser?Phe?Pro?Phe?Glu?Lys?Asn

755 760 765

Cys?Glu?Gly?Asn?Leu?Gly?Val?Ser?Phe?Asn?Phe?Ser?Gly?Leu?Gln?Val

770 775 780

Leu?Glu?Val?Gly?Ser?Ser?Pro?Glu?Leu?Thr?Val?Thr?Val?Thr?Val?Trp

785 790 795 800

Asn?Glu?Gly?Glu?Asp?Ser?Tyr?Gly?Thr?Leu?Ile?Lys?Phe?Tyr?Tyr?Pro

805 810 815

Ala?Glu?Leu?Ser?Tyr?Arg?Arg?Val?Thr?Arg?Ala?Gln?Gln?Pro?His?Pro

820 825 830

Tyr?Pro?Leu?Arg?Leu?Ala?Cys?Glu?Ala?Glu?Pro?Thr?Gly?Gln?Glu?Ser

835 840 845

Leu?Arg?Ser?Ser?Ser?Cys?Ser?Ile?Asn?His?Pro?Ile?Phe?Arg?Glu?Gly

850 855 860

Ala?Lys?Ala?Thr?Phe?Met?Ile?Thr?Phe?Asp?Val?Ser?Tyr?Lys?Ala?Phe

865 870 875 880

Leu?Gly?Asp?Arg?Leu?Leu?Leu?Arg?Ala?Ser?Ala?Ser?Ser?Glu?Asn?Asn

885 890 895

Lys?Pro?Glu?Thr?Ser?Lys?Thr?Ala?Phe?Gln?Leu?Glu?Leu?Pro?Val?Lys

900 905 910

Tyr?Thr?Val?Tyr?Thr?Val?Ile?Ser?Arg?Gln?Glu?Asp?Ser?Thr?Lys?His

915 920 925

Phe?Asn?Phe?Ser?Ser?Ser?His?Gly?Glu?Arg?Gln?Lys?Glu?Ala?Glu?His

930 935 940

Arg?Tyr?Arg?Val?Asn?Asn?Leu?Ser?Pro?Leu?Thr?Leu?Ala?Ile?Ser?Val

945 950 955 960

Asn?Phe?Trp?Val?Pro?Ile?Leu?Leu?Asn?Gly?Val?Ala?Val?Trp?Asp?Val

965 970 975

Thr?Leu?Arg?Ser?Pro?Ala?Gln?Gly?Val?Ser?Cys?Val?Ser?Gln?Arg?Glu

980 985 990

Pro?Pro?Gln?His?Ser?Asp?Leu?Leu?Thr?Gln?Ile?Gln?Gly?Arg?Ser?Val

995 1000 1005

Leu?Asp?Cys?Ala?Ile?Ala?Asp?Cys?Leu?His?Leu?Arg?Cys?Asp?Ile?Pro

1010 1015 1020

Ser?Leu?Gly?Thr?Leu?Asp?Glu?Leu?Asp?Phe?Ile?Leu?Lys?Gly?Asn?Leu

1025 1030 1035 1040

Ser?Phe?Gly?Trp?Ile?Ser?Gln?Thr?Leu?Gln?Lys?Lys?Val?Leu?Leu?Leu

1045 1050 1055

Ser?Glu?Ala?Glu?Ile?Thr?Phe?Asn?Thr?Ser?Val?Tyr?Ser?Gln?Leu?Pro

1060 1065 1070

Gly?Gln?Glu?Ala?Phe?Leu?Arg?Ala?Gln?Val?Ser?Thr?Met?Leu?Glu?Glu

1075 1080 1085

Tyr?Val?Val?Tyr?Glu?Pro?Val?Phe?Leu?Met?Val?Phe?Ser?Ser?Val?Gly

1090 1095 1100

Gly?Leu?Leu?Leu?Leu?Ala?Leu?Ile?Thr?Val?Ala?Leu?Tyr?Lys?Leu?Gly

1105 1110 1115 1120

Phe?Phe?Lys?Arg?Gln?Tyr?Lys?Glu?Met?Leu?Asp?Leu?Pro?Ser?Ala?Asp

1125 1130 1135

Pro?Asp?Pro?Ala?Gly?Gln?Ala?Asp?Ser?Asn?His?Glu?Thr?Pro?Pro?His

1140 1145 1150

Leu?Thr?Ser

115

<210>47

<211>49

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>47

agttacggat?ccggcaccat?gaccttcggc?actgtgatcc?tcctgtgtg 49

<210>48

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>48

gctggacgat?ggcatccac 19

<210>49

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>49

gtagagttac?ggatccggca?ccat 24

<210>50

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>50

gcagccagct?tcggacagac 20

<210>51

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>51

ccatgtccac?agaacagaga?g 21

<210>52

<211>3803

<212>DNA

＜213〉mice

<220>

<221>CDS

<222>(1)..(3483)

<220>

＜223〉artificial sequence note: primer

<400>52

atg?gtc?cgt?gga?gtt?gtg?atc?ctc?ctg?tgt?ggc?tgg?gcc?ctg?gct?tcc 48

Met?Val?Arg?Gly?Val?Val?Ile?Leu?Leu?Cys?Gly?Trp?Ala?Leu?Ala?Ser

1 5 10 15

tgt?cat?ggg?tct?aac?ctg?gat?gtg?gag?aag?ccc?gtc?gtg?ttc?aaa?gag 96

Cys?His?Gly?Ser?Asn?Leu?Asp?Val?Glu?Lys?Pro?Val?Val?Phe?Lys?Glu

20 25 30

gat?gca?gcc?agc?ttc?gga?cag?act?gtg?gtg?cag?ttt?ggt?gga?tct?cga 144

Asp?Ala?Ala?Ser?Phe?Gly?Gln?Thr?Val?Val?Gln?Phe?Gly?Gly?Ser?Arg

35 40 45

ctc?gtg?gtg?gga?gcc?cct?ctg?gag?gcg?gtg?gca?gtc?aac?caa?aca?gga 192

Leu?Val?Val?Gly?Ala?Pro?Leu?Glu?Ala?Val?Ala?Val?Asn?Gln?Thr?Gly

50 55 60

cag?tcg?tct?gac?tgt?ccg?cct?gcc?act?ggc?gtg?tgc?cag?ccc?atc?tta 240

Gln?Ser?Ser?Asp?Cys?Pro?Pro?Ala?Thr?Gly?Val?Cys?Gln?Pro?Ile?Leu

65 70 75 80

ctg?cac?att?ccc?cta?gag?gca?gtg?aac?atg?tcc?ctg?ggc?ctg?tct?ctg 288

Leu?His?Ile?Pro?Leu?Glu?Ala?Val?Asn?Met?Ser?Leu?Gly?Leu?Ser?Leu

85 90 95

gtg?gct?gac?acc?aat?aac?tcc?cag?ttg?ctg?gct?tgt?ggt?cca?act?gca 336

Val?Ala?Asp?Thr?Asn?Asn?Ser?Gln?Leu?Leu?Ala?Cys?Gly?Pro?Thr?Ala

100 105 110

cag?aga?gct?tgt?gca?aag?aac?atg?tat?gca?aaa?ggt?tcc?tgc?ctc?ctt 384

Gln?Arg?Ala?Cys?Ala?Lys?Asn?Met?Tyr?Ala?Lys?Gly?Ser?Cys?Leu?Leu

115 120 125

ctg?ggc?tcc?agc?ttg?cag?ttc?atc?cag?gca?atc?cct?gct?acc?atg?cca 432

Leu?Gly?Ser?Ser?Leu?Gln?Phe?Ile?Gln?Ala?Ile?Pro?Ala?Thr?Met?Pro

130 135 140

gag?tgt?cca?gga?caa?gag?atg?gac?att?gct?ttc?ctg?att?gat?ggc?tcc 480

Glu?Cys?Pro?Gly?Gln?Glu?Met?Asp?Ile?Ala?Phe?Leu?Ile?Asp?Gly?Ser

145 150 155 160

ggc?agc?att?gat?caa?agt?gac?ttt?acc?cag?atg?aag?gac?ttc?gtc?aaa 528

Gly?Ser?Ile?Asp?Gln?Ser?Asp?Phe?Thr?Gln?Met?Lys?Asp?Phe?Val?Lys

165 170 175

gct?ttg?atg?ggc?cag?ttg?gcg?agc?acc?agc?acc?tcg?ttc?tcc?ctg?atg 576

Ala?Leu?Met?Gly?Gln?Leu?Ala?Ser?Thr?Ser?Thr?Ser?Phe?Ser?Leu?Met

180 185 190

caa?tac?tca?aac?atc?ctg?aag?act?cat?ttt?acc?ttc?acg?gaa?ttc?aag 624

Gln?Tyr?Ser?Asn?Ile?Leu?Lys?Thr?His?Phe?Thr?Phe?Thr?Glu?Phe?Lys

195 200 205

agc?agc?ctg?agc?cct?cag?agc?ctg?gtg?gat?gcc?atc?gtc?cag?ctc?caa 672

Ser?Ser?Leu?Ser?Pro?Gln?Ser?Leu?Val?Asp?Ala?Ile?Val?Gln?Leu?Gln

210 215 220

ggc?ctg?acg?tac?aca?gcc?tcg?ggc?atc?cag?aaa?gtg?gtg?aaa?gag?cta 720

Gly?Leu?Thr?Tyr?Thr?Ala?Ser?Gly?Ile?Gln?Lys?Val?Val?Lys?Glu?Leu

225 230 235 240

ttt?cat?agc?aag?aat?ggg?gcc?cga?aaa?agt?gcc?aag?aag?ata?cta?att 768

Phe?His?Ser?Lys?Asn?Gly?Ala?Arg?Lys?Ser?Ala?Lys?Lys?Ile?Leu?Ile

245 250 255

gtc?atc?aca?gat?ggg?cag?aaa?ttc?aga?gac?ccc?ctg?gag?tat?aga?cat 816

Val?Ile?Thr?Asp?Gly?Gln?Lys?Phe?Arg?Asp?Pro?Leu?Glu?Tyr?Arg?His

260 265 270

gtc?atc?cct?gaa?gca?gag?aaa?gct?ggg?atc?att?cgc?tat?gct?ata?ggg 864

Val?Ile?Pro?Glu?Ala?Glu?Lys?Ala?Gly?Ile?Ile?Arg?Tyr?Ala?Ile?Gly

275 280 285

gtg?gga?gat?gcc?ttc?cgg?gaa?ccc?act?gcc?cta?cag?gag?ctg?aac?acc 912

Val?Gly?Asp?Ala?Phe?Arg?Glu?Pro?Thr?Ala?Leu?Gln?Glu?Leu?Asn?Thr

290 295 300

att?ggc?tca?gct?ccc?tcg?cag?gac?cac?gtg?ttc?aag?gtg?ggc?aat?ttt 960

Ile?Gly?Ser?Ala?Pro?Ser?Gln?Asp?His?Val?Phe?Lys?Val?Gly?Asn?Phe

305 310 315 320

gta?gca?ctt?cgc?agc?atc?cag?cgg?caa?att?cag?gag?aaa?atc?ttt?gcc 1008

Val?Ala?Leu?Arg?Ser?Ile?Gln?Arg?Gln?Ile?Gln?Glu?Lys?Ile?Phe?Ala

325 330 335

att?gaa?gga?acc?gaa?tca?agg?tca?agt?agt?tcc?ttt?cag?cac?gag?atg 1056

Ile?Glu?Gly?Thr?Glu?Ser?Arg?Ser?Ser?Ser?Ser?Phe?Gln?His?Glu?Met

340 345 350

tca?caa?gaa?ggt?ttc?agc?tca?gct?ctc?tca?atg?gat?gga?cca?gtt?ctg 1104

Ser?Gln?Glu?Gly?Phe?Ser?Ser?Ala?Leu?Ser?Met?Asp?Gly?Pro?Val?Leu

355 360 365

ggg?gct?gtg?gga?ggc?ttc?agc?tgg?tct?gga?ggt?gcc?ttc?ttg?tac?ccc 1152

Gly?Ala?Val?Gly?Gly?Phe?Ser?Trp?Ser?Gly?Gly?Ala?Phe?Leu?Tyr?Pro

370 375 380

tca?aat?atg?aga?tcc?acc?ttc?atc?aac?atg?tct?cag?gag?aac?gag?gat 1200

Ser?Asn?Met?Arg?Ser?Thr?Phe?Ile?Asn?Met?Ser?Gln?Glu?Asn?Glu?Asp

385 390 395 400

atg?agg?gac?gct?tac?ctg?ggt?tac?tcc?acc?gca?ctg?gcc?ttt?tgg?aag 1248

Met?Arg?Asp?Ala?Tyr?Leu?Gly?Tyr?Ser?Thr?Ala?Leu?Ala?Phe?Trp?Lys

405 410 415

ggg?gtc?cac?agc?ctg?atc?ctg?ggg?gcc?cct?cgc?cac?cag?cac?acg?ggg 1296

Gly?Val?His?Ser?Leu?Ile?Leu?Gly?Ala?Pro?Arg?His?Gln?His?Thr?Gly

420 425 430

aag?gtt?gtc?atc?ttt?acc?cag?gaa?tcc?agg?cac?tgg?agg?ccc?aag?tct 1344

Lys?Val?Val?Ile?Phe?Thr?Gln?Glu?Ser?Arg?His?Trp?Arg?Pro?Lys?Ser

435 440 445

gaa?gtc?aga?ggg?aca?cag?atc?ggc?tcc?tac?ttt?ggg?gca?tct?ctc?tgt 1392

Glu?Val?Arg?Gly?Thr?Gln?Ile?Gly?Ser?Tyr?Phe?Gly?Ala?Ser?Leu?Cys

450 455 460

tct?gtg?gac?atg?gat?aga?gat?ggc?agc?act?gac?ctg?gtc?ctg?att?gga 1440

Ser?Val?Asp?Met?Asp?Arg?Asp?Gly?Ser?Thr?Asp?Leu?Val?Leu?Ile?Gly

465 470 475 480

gtc?ccc?cat?tac?tat?gag?cac?acc?cga?ggg?ggg?cag?gtg?tcg?gtg?tgc 1488

Val?Pro?His?Tyr?Tyr?Glu?His?Thr?Arg?Gly?Gly?Gln?Val?Ser?Val?Cys

485 490 495

ccc?atg?cct?ggt?gtg?agg?agc?agg?tgg?cat?tgt?ggg?acc?acc?ctc?cat 1536

Pro?Met?Pro?Gly?Val?Arg?Ser?Arg?Trp?His?Cys?Gly?Thr?Thr?Leu?His

500 505 510

ggg?gag?cag?ggc?cat?cct?tgg?ggc?cgc?ttt?ggg?gcg?gct?ctg?aca?gtg 1584

Gly?Glu?Gln?Gly?His?Pro?Trp?Gly?Arg?Phe?Gly?Ala?Ala?Leu?Thr?Val

515 520 525

cta?ggg?gac?gtg?aat?ggg?gac?agt?ctg?gcg?gat?gtg?gct?att?ggt?gca 1632

Leu?Gly?Asp?Val?Asn?Gly?Asp?Ser?Leu?Ala?Asp?Val?Ala?Ile?Gly?Ala

530 535 540

ccc?gga?gag?gag?gag?aac?aga?ggt?gct?gtc?tac?ata?ttt?cat?gga?gcc 1680

Pro?Gly?Glu?Glu?Glu?Asn?Arg?Gly?Ala?Val?Tyr?Ile?Phe?His?Gly?Ala

545 550 555 560

tcg?aga?cag?gac?atc?gct?ccc?tcg?cct?agc?cag?cgg?gtc?act?ggc?tcc 1728

Ser?Arg?Gln?Asp?Ile?Ala?Pro?Ser?Pro?Ser?Gln?Arg?Val?Thr?Gly?Ser

565 570 575

cag?ctc?ttc?ctg?agg?ctc?caa?tat?ttt?ggg?cag?tca?tta?agt?ggg?ggt 1776

Gln?Leu?Phe?Leu?Arg?Leu?Gln?Tyr?Phe?Gly?Gln?Ser?Leu?Ser?Gly?Gly

580 585 590

cag?gac?ctt?aca?cag?gat?ggc?ctg?gtg?gac?ctg?gcc?gtg?gga?gcc?cag 1824

Gln?Asp?Leu?Thr?Gln?Asp?Gly?Leu?Val?Asp?Leu?Ala?Val?Gly?Ala?Gln

595 600 605

ggg?cac?gtg?ctg?ctg?ctt?agg?agt?ctg?cct?ttg?ctg?aaa?gtg?ggg?atc 1872

Gly?His?Val?Leu?Leu?Leu?Arg?Ser?Leu?Pro?Leu?Leu?Lys?Val?Gly?Ile

610 615 620

tcc?att?aga?ttt?gcc?ccc?tca?gag?gtg?gca?aag?act?gtg?tac?cag?tgc 1920

Ser?Ile?Arg?Phe?Ala?Pro?Ser?Glu?Val?Ala?Lys?Thr?Val?Tyr?Gln?Cys

625 630 635 640

tgg?gga?agg?act?ccc?act?gtc?ctc?gaa?gct?gga?gag?gcc?acc?gtc?tgt 1968

Trp?Gly?Arg?Thr?Pro?Thr?Val?Leu?Glu?Ala?Gly?Glu?Ala?Thr?Val?Cys

645 650 655

ctc?act?gtc?cgc?aaa?ggt?tca?cct?gac?ctg?tta?ggt?gat?gtc?caa?agc 2016

Leu?Thr?Val?Arg?Lys?Gly?Ser?Pro?Asp?Leu?Leu?Gly?Asp?Val?Gln?Ser

660 665 670

tct?gtc?agg?tat?gat?ctg?gcg?ttg?gat?ccg?ggc?cgt?ctg?att?tct?cgt 2064

Ser?Val?Arg?Tyr?Asp?Leu?Ala?Leu?Asp?Pro?Gly?Arg?Leu?Ile?Ser?Arg

675 680 685

gcc?att?ttt?gat?gag?acg?aag?aac?tgc?act?ttg?acc?cga?agg?aag?act 2112

Ala?Ile?Phe?Asp?Glu?Thr?Lys?Asn?Cys?Thr?Leu?Thr?Arg?Arg?Lys?Thr

690 695 700

ctg?ggg?ctt?ggt?gat?cac?tgc?gaa?aca?atg?aag?ctg?ctt?ttg?cca?gac 2160

Leu?Gly?Leu?Gly?Asp?His?Cys?Glu?Thr?Met?Lys?Leu?Leu?Leu?Pro?Asp

705 710 715 720

tgt?gtg?gag?gat?gca?gtg?acc?cct?atc?atc?ctg?cgc?ctt?aac?tta?tcc 2208

Cys?Val?Glu?Asp?Ala?Val?Thr?Pro?Ile?Ile?Leu?Arg?Leu?Asn?Leu?Ser

725 730 735

ctg?gca?ggg?gac?tct?gct?cca?tcc?agg?aac?ctt?cgt?cct?gtg?ctg?gct 2256

Leu?Ala?Gly?Asp?Ser?Ala?Pro?Ser?Arg?Asn?Leu?Arg?Pro?Val?Leu?Ala

740 745 750

gtg?ggc?tca?caa?gac?cat?gta?aca?gct?tct?ttc?ccg?ttt?gag?aag?aac 2304

Val?Gly?Ser?Gln?Asp?His?Val?Thr?Ala?Ser?Phe?Pro?Phe?Glu?Lys?Asn

755 760 765

tgt?aag?cag?gag?ctc?ctg?tgt?gag?ggg?agc?ctg?ggc?gtc?agc?ttc?aac 2352

Cys?Lys?Gln?Glu?Leu?Leu?Cys?Glu?Gly?Asn?Leu?Gly?Val?Ser?Phe?Asn

770 775 780

ttc?tca?ggc?ctg?cag?gtc?ttg?gag?gta?gga?agc?tcc?cca?gag?ctc?act 2400

Phe?Ser?Gly?Leu?Gln?Val?Leu?Glu?Val?Gly?Ser?Ser?Pro?Glu?Leu?Thr

785 790 795 800

gtg?aca?gta?aca?gtt?tgg?aat?gag?ggt?gag?gac?agc?tat?gga?acc?tta 2448

Val?Thr?Val?Thr?Val?Trp?Asn?Glu?Gly?Glu?Asp?Ser?Tyr?Gly?Thr?Leu

805 810 815

atc?aag?ttc?tac?tac?cca?gca?gag?cta?tct?tac?cga?cgg?gtg?aca?aga 2496

Ile?Lys?Phe?Tyr?Tyr?Pro?Ala?Glu?Leu?Ser?Tyr?Arg?Arg?Val?Thr?Arg

820 825 830

gcc?cag?caa?cct?cat?ccg?tac?cca?cta?cgc?ctg?gca?tgt?gag?gct?gag 2544

Ala?Gln?Gln?Pro?His?Pro?Tyr?Pro?Leu?Arg?Leu?Ala?Cys?Glu?Ala?Glu

835 840 845

ccc?acg?ggc?cag?gag?agc?ctg?agg?agc?agc?agc?tgt?agc?atc?aat?cac 2592

Pro?Thr?Gly?Gln?Glu?Ser?Leu?Arg?Ser?Ser?Ser?Cys?Ser?Ile?Asn?His

850 855 860

ccc?atc?ttc?cga?gaa?ggt?gcc?aag?gcc?acc?ttc?atg?atc?aca?ttt?gat 2640

Pro?Ile?Phe?Arg?Glu?Gly?Ala?Lys?Ala?Thr?Phe?Met?Ile?Thr?Phe?Asp

865 870 875 880

gtc?tcc?tac?aag?gcc?ttc?ctg?gga?gac?agg?ttg?ctt?ctg?agg?gcc?agc 2688

Val?Ser?Tyr?Lys?Ala?Phe?Leu?Gly?Asp?Arg?Leu?Leu?Leu?Arg?Ala?Ser

885 890 895

gca?agc?agt?gag?aat?aat?aag?cct?gaa?acc?agc?aag?act?gcc?ttc?cag 2736

Ala?Ser?Ser?Glu?Asn?Asn?Lys?Pro?Glu?Thr?Ser?Lys?Thr?Ala?Phe?Gln

900 905 910

ctg?gag?ctt?ccg?gtg?aag?tac?acg?gtc?tat?acc?gtg?atc?agt?agg?cag 2784

Leu?Glu?Leu?Pro?Val?Lys?Tyr?Thr?Val?Tyr?Thr?Val?Ile?Ser?Arg?Gln

915 920 925

gaa?gat?tct?acc?aag?cat?ttc?aac?ttc?tca?tct?tcc?cac?ggg?gag?aga 2832

Glu?Asp?Ser?Thr?Lys?His?Phe?Asn?Phe?Ser?Ser?Ser?His?Gly?Glu?Arg

930 935 940

cag?aaa?gag?gcc?gaa?cat?cga?tat?cgt?gtg?aat?aac?ctg?agt?cca?ttg 2880

Gln?Lys?Glu?Ala?Glu?His?Arg?Tyr?Arg?Val?Asn?Asn?Leu?Ser?Pro?Leu

945 950 955 960

acg?ctg?gcc?atc?agc?gtt?aac?ttc?tgg?gtc?ccc?atc?ctt?ctg?aat?ggt 2928

Thr?Leu?Ala?Ile?Ser?Val?Asn?Phe?Trp?Val?Pro?Ile?Leu?Leu?Asn?Gly

965 970 975

gtg?gcc?gtg?tgg?gat?gtg?act?ctg?agg?agc?cca?gca?cag?ggt?gtc?tcc 2976

Val?Ala?Val?Trp?Asp?Val?Thr?Leu?Arg?Ser?Pro?Ala?Gln?Gly?Val?Ser

980 985 990

tgt?gtg?tca?cag?agg?gaa?cct?cct?caa?cat?tcc?gac?ctt?ctg?acc?cag 3024

Cys?Val?Ser?Gln?Arg?Glu?Pro?Pro?Gln?His?Ser?Asp?Leu?Leu?Thr?Gln

995 1000 1005

atc?caa?gga?cgc?tct?gtg?ctg?gac?tgc?gcc?atc?gcc?gac?tgc?ctg?cac 3072

Ile?Gln?Gly?Arg?Ser?Val?Leu?Asp?Cys?Ala?Ile?Ala?Asp?Cys?Leu?His

1010 1015 1020

ctc?cgc?tgt?gac?atc?ccc?tcc?ttg?ggc?acc?ctg?gat?gag?ctt?gac?ttc 3120

Leu?Arg?Cys?Asp?Ile?Pro?Ser?Leu?Gly?Thr?Leu?Asp?Glu?Leu?Asp?Phe

1025 1030 1035 1040

att?ctg?aag?ggc?aac?ctc?agc?ttc?ggc?tgg?atc?agt?cag?aca?ttg?cag 3168

Ile?Leu?Lys?Gly?Asn?Leu?Ser?Phe?Gly?Trp?Ile?Ser?Gln?Thr?Leu?Gln

1045 1050 1055

aaa?aag?gtg?ttg?ctc?ctg?agt?gag?gct?gaa?atc?aca?ttc?aac?aca?tct 3216

Lys?Lys?Val?Leu?Leu?Leu?Ser?Glu?Ala?Glu?Ile?Thr?Phe?Asn?Thr?Ser

1060 1065 1070

gtg?tat?tcc?cag?ctg?ccg?gga?cag?gag?gca?ttt?ctg?aga?gcc?cag?gtg 3264

Val?Tyr?Ser?Gln?Leu?Pro?Gly?Gln?Glu?Ala?Phe?Leu?Arg?Ala?Gln?Val

1075 1080 1085

tca?acg?atg?cta?gaa?gaa?tac?gtg?gtc?tat?gag?ccc?gtc?ttc?ctc?atg 3312

Ser?Thr?Met?Leu?Glu?Glu?Tyr?Val?Val?Tyr?Glu?Pro?Val?Phe?Leu?Met

1090 1095 1100

gtg?ttc?agc?tca?gtg?gga?ggt?ctg?ctg?tta?ctg?gct?ctc?atc?act?gtg 3360

Val?Phe?Ser?Ser?Val?Gly?Gly?Leu?Leu?Leu?Leu?Ala?Leu?Ile?Thr?Val

1105 1110 1115 1120

gcg?ctg?tac?aag?ctt?ggc?ttc?ttc?aaa?cgt?cag?tat?aaa?gag?atg?ctg 3408

Ala?Leu?Tyr?Lys?Leu?Gly?Phe?Phe?Lys?Arg?Gln?Tyr?Lys?Glu?Met?Leu

1125 1130 1135

gat?cta?cca?tct?gca?gat?cct?gac?cca?gcc?ggc?cag?gca?gat?tcc?aac 3456

Asp?Leu?Pro?Ser?Ala?Asp?Pro?Asp?Pro?Ala?Gly?Gln?Ala?Asp?Ser?Asn

1140 1145 1150

cat?gag?act?cct?cca?cat?ctc?acg?tcc?taggaatcta?ctttcctgta 3503

His?Glu?Thr?Pro?Pro?His?Leu?Thr?Ser

1155 1160

tatctccaca?attacgagat?tggttttgct?tttgcctatg?aatctactgg?catgggaaca 3563

agttctcttc?agctctgggc?tagcctggga?aacttcccag?aaatgatgcc?ctacctcctg?3623

agctgggaga?tttttatggt?ttgcccatgt?gtcagatttc?agtgctgatc?cacttttttt?3683

gcaagagcag?gaatggggtc?agcataaatt?tacatatgga?taagaactaa?cacaagactg?3743

agtaatatgc?tcaatattca?atgtattgct?tgtataaatt?tttaaaaaat?aaaatgaaan?3803

<210>53

<211>1161

<212>PRT

＜213〉mice

<400>53

Met?Val?Arg?Gly?Val?Val?Ile?Leu?Leu?Cys?Gly?Trp?Ala?Leu?Ala?Ser

1 5 10 15

Cys?His?Gly?Ser?Asn?Leu?Asp?Val?Glu?Lys?Pro?Val?Val?Phe?Lys?Glu

20 25 30

Asp?Ala?Ala?Ser?Phe?Gly?Gln?Thr?Val?Val?Gln?Phe?Gly?Gly?Ser?Arg

35 40 45

Leu?Val?Val?Gly?Ala?Pro?Leu?Glu?Ala?Val?Ala?Val?Asn?Gln?Thr?Gly

50 55 60

Gln?Ser?Ser?Asp?Cys?Pro?Pro?Ala?Thr?Gly?Val?Cys?Gln?Pro?Ile?Leu

65 70 75 80

Leu?His?Ile?Pro?Leu?Glu?Ala?Val?Asn?Met?Ser?Leu?Gly?Leu?Ser?Leu

85 90 95

Val?Ala?Asp?Thr?Asn?Asn?Ser?Gln?Leu?Leu?Ala?Cys?Gly?Pro?Thr?Ala

100 105 110

Gln?Arg?Ala?Cys?Ala?Lys?Asn?Met?Tyr?Ala?Lys?Gly?Ser?Cys?Leu?Leu

115 120 125

Leu?Gly?Ser?Ser?Leu?Gln?Phe?Ile?Gln?Ala?Ile?Pro?Ala?Thr?Met?Pro

130 135 140

Glu?Cys?Pro?Gly?Gln?Glu?Met?Asp?Ile?Ala?Phe?Leu?Ile?Asp?Gly?Ser

145 150 155 160

Gly?Ser?Ile?Asp?Gln?Ser?Asp?Phe?Thr?Gln?Met?Lys?Asp?Phe?Val?Lys

165 170 175

Ala?Leu?Met?Gly?Gln?Leu?Ala?Ser?Thr?Ser?Thr?Ser?Phe?Ser?Leu?Met

180 185 190

Gln?Tyr?Ser?Asn?Ile?Leu?Lys?Thr?His?Phe?Thr?Phe?Thr?Glu?Phe?Lys

195 200 205

Ser?Ser?Leu?Ser?Pro?Gln?Ser?Leu?Val?Asp?Ala?Ile?Val?Gln?Leu?Gln

210 215 220

Gly?Leu?Thr?Tyr?Thr?Ala?Ser?Gly?Ile?Gln?Lys?Val?Val?Lys?Glu?Leu

225 230 235 240

Phe?His?Ser?Lys?Asn?Gly?Ala?Arg?Lys?Ser?Ala?Lys?Lys?Ile?Leu?Ile

245 250 255

Val?Ile?Thr?Asp?Gly?Gln?Lys?Phe?Arg?Asp?Pro?Leu?Glu?Tyr?Arg?His

260 265 270

Val?Ile?Pro?Glu?Ala?Glu?Lys?Ala?Gly?Ile?Ile?Arg?Tyr?Ala?Ile?Gly

275 280 285

Val?Gly?Asp?Ala?Phe?Arg?Glu?Pro?Thr?Ala?Leu?Gln?Glu?Leu?Asn?Thr

290 295 300

Ile?Gly?Ser?Ala?Pro?Ser?Gln?Asp?His?Val?Phe?Lys?Val?Gly?Asn?Phe

305 310 315 320

Val?Ala?Leu?Arg?Ser?Ile?Gln?Arg?Gln?Ile?Gln?Glu?Lys?Ile?Phe?Ala

325 330 335

Ile?Glu?Gly?Thr?Glu?Ser?Arg?Ser?Ser?Ser?Ser?Phe?Gln?His?Glu?Met

340 345 350

Ser?Gln?Glu?Gly?Phe?Ser?Ser?Ala?Leu?Ser?Met?Asp?Gly?Pro?Val?Leu

355 360 365

Gly?Ala?Val?Gly?Gly?Phe?Ser?Trp?Ser?Gly?Gly?Ala?Phe?Leu?Tyr?Pro

370 375 380

Ser?Asn?Met?Arg?Ser?Thr?Phe?Ile?Asn?Met?Ser?Gln?Glu?Asn?Glu?Asp

385 390 395 400

Met?Arg?Asp?Ala?Tyr?Leu?Gly?Tyr?Ser?Thr?Ala?Leu?Ala?Phe?Trp?Lys

405 410 415

Gly?Val?His?Ser?Leu?Ile?Leu?Gly?Ala?Pro?Arg?His?Gln?His?Thr?Gly

420 425 430

Lys?Val?Val?Ile?Phe?Thr?Gln?Glu?Ser?Arg?His?Trp?Arg?Pro?Lys?Ser

435 440 445

Glu?Val?Arg?Gly?Thr?Gln?Ile?Gly?Ser?Tyr?Phe?Gly?Ala?Ser?Leu?Cys

450 455 460

Ser?Val?Asp?Met?Asp?Arg?Asp?Gly?Ser?Thr?Asp?Leu?Val?Leu?Ile?Gly

465 470 475 480

Val?Pro?His?Tyr?Tyr?Glu?His?Thr?Arg?Gly?Gly?Gln?Val?Ser?Val?Cys

485 490 495

Pro?Met?Pro?Gly?Val?Arg?Ser?Arg?Trp?His?Cys?Gly?Thr?Thr?Leu?His

500 505 510

Gly?Glu?Gln?Gly?His?Pro?Trp?Gly?Arg?Phe?Gly?Ala?Ala?Leu?Thr?Val

515 520 525

Leu?Gly?Asp?Val?Asn?Gly?Asp?Ser?Leu?Ala?Asp?Val?Ala?Ile?Gly?Ala

530 535 540

Pro?Gly?Glu?Glu?Glu?Asn?Arg?Gly?Ala?Val?Tyr?Ile?Phe?His?Gly?Ala

545 550 555 560

Ser?Arg?Gln?Asp?Ile?Ala?Pro?Ser?Pro?Ser?Gln?Arg?Val?Thr?Gly?Ser

565 570 575

Gln?Leu?Phe?Leu?Arg?Leu?Gln?Tyr?Phe?Gly?Gln?Ser?Leu?Ser?Gly?Gly

580 585 590

Gln?Asp?Leu?Thr?Gln?Asp?Gly?Leu?Val?Asp?Leu?Ala?Val?Gly?Ala?Gln

595 600 605

Gly?His?Val?Leu?Leu?Leu?Arg?Ser?Leu?Pro?Leu?Leu?Lys?Val?Gly?Ile

610 615 620

Ser?Ile?Arg?Phe?Ala?Pro?Ser?Glu?Val?Ala?Lys?Thr?Val?Tyr?Gln?Cys

625 630 635 640

Trp?Gly?Arg?Thr?Pro?Thr?Val?Leu?Glu?Ala?Gly?Glu?Ala?Thr?Val?Cys

645 650 655

Leu?Thr?Val?Arg?Lys?Gly?Ser?Pro?Asp?Leu?Leu?Gly?Asp?Val?Gln?Ser

660 665 670

Ser?Val?Arg?Tyr?Asp?Leu?Ala?Leu?Asp?Pro?Gly?Arg?Leu?Ile?Ser?Arg

675 680 685

Ala?Ile?Phe?Asp?Glu?Thr?Lys?Asn?Cys?Thr?Leu?Thr?Arg?Arg?Lys?Thr

690 695 700

Leu?Gly?Leu?Gly?Asp?His?Cys?Glu?Thr?Met?Lys?Leu?Leu?Leu?Pro?Asp

705 710 715 720

Cys?Val?Glu?Asp?Ala?Val?Thr?Pro?Ile?Ile?Leu?Arg?Leu?Asn?Leu?Ser

725 730 735

Leu?Ala?Gly?Asp?Ser?Ala?Pro?Ser?Arg?Asn?Leu?Arg?Pro?Val?Leu?Ala

740 745 750

Val?Gly?Ser?Gln?Asp?His?Val?Thr?Ala?Ser?Phe?Pro?Phe?Glu?Lys?Asn

755 760 765

Cys?Lys?Gln?Glu?Leu?Leu?Cys?Glu?Gly?Asn?Leu?Gly?Val?Ser?Phe?Asn

770 775 780

Phe?Ser?Gly?Leu?Gln?Val?Leu?Glu?Val?Gly?Ser?Ser?Pro?Glu?Leu?Thr

785 790 795 800

Val?Thr?Val?Thr?Val?Trp?Asn?Glu?Gly?Glu?Asp?Ser?Tyr?Gly?Thr?Leu

805 810 815

Ile?Lys?Phe?Tyr?Tyr?Pro?Ala?Glu?Leu?Ser?Tyr?Arg?Arg?Val?Thr?Arg

820 825 830

Ala?Gln?Gln?Pro?His?Pro?Tyr?Pro?Leu?Arg?Leu?Ala?Cys?Glu?Ala?Glu

835 840 845

Pro?Thr?Gly?Gln?Glu?Ser?Leu?Arg?Ser?Ser?Ser?Cys?Ser?Ile?Asn?His

850 855 860

Pro?Ile?Phe?Arg?Glu?Gly?Ala?Lys?Ala?Thr?Phe?Met?Ile?Thr?Phe?Asp

865 870 875 880

Val?Ser?Tyr?Lys?Ala?Phe?Leu?Gly?Asp?Arg?Leu?Leu?Leu?Arg?Ala?Ser

885 890 895

Ala?Ser?Ser?Glu?Asn?Asn?Lys?Pro?Glu?Thr?Ser?Lys?Thr?Ala?Phe?Gln

900 905 910

Leu?Glu?Leu?Pro?Val?Lys?Tyr?Thr?Val?Tyr?Thr?Val?Ile?Ser?Arg?Gln

915 920 925

Glu?Asp?Ser?Thr?Lys?His?Phe?Asn?Phe?Ser?Ser?Ser?His?Gly?Glu?Arg

930 935 940

Gln?Lys?Glu?Ala?Glu?His?Arg?Tyr?Arg?Val?Asn?Asn?Leu?Ser?Pro?Leu

945 950 955 960

Thr?Leu?Ala?Ile?Ser?Val?Asn?Phe?Trp?Val?Pro?Ile?Leu?Leu?Asn?Gly

965 970 975

Val?Ala?Val?Trp?Asp?Val?Thr?Leu?Arg?Ser?Pro?Ala?Gln?Gly?Val?Ser

980 985 990

Cys?Val?Ser?Gln?Arg?Glu?Pro?Pro?Gln?His?Ser?Asp?Leu?Leu?Thr?Gln

995 1000 1005

Ile?Gln?Gly?Arg?Ser?Val?Leu?Asp?Cys?Ala?Ile?Ala?Asp?Cys?Leu?His

1010 1015 1020

Leu?Arg?Cys?Asp?Ile?Pro?Ser?Leu?Gly?Thr?Leu?Asp?Glu?Leu?Asp?Phe

1025 1030 1035 1040

Ile?Leu?Lys?Gly?Asn?Leu?Ser?Phe?Gly?Trp?Ile?Ser?Gln?Thr?Leu?Gln

1045 1050 1055

Lys?Lys?Val?Leu?Leu?Leu?Ser?Glu?Ala?Glu?Ile?Thr?Phe?Asn?Thr?Ser

1060 1065 1070

Val?Tyr?Ser?Gln?Leu?Pro?Gly?Gln?Glu?Ala?Phe?Leu?Arg?Ala?Gln?Val

1075 1080 1085

Ser?Thr?Met?Leu?Glu?Glu?Tyr?Val?Val?Tyr?Glu?Pro?Val?Phe?Leu?Met

1090 1095 1100

Val?Phe?Ser?Ser?Val?Gly?Gly?Leu?Leu?Leu?Leu?Ala?Leu?Ile?Thr?Val

1105 1110 1115 1120

Ala?Leu?Tyr?Lys?Leu?Gly?Phe?Phe?Lys?Arg?Gln?Tyr?Lys?Glu?Met?Leu

1125 1130 1135

Asp?Leu?Pro?Ser?Ala?Asp?Pro?Asp?Pro?Ala?Gly?Gln?Ala?Asp?Ser?Asn

1140 1145 1150

His?Glu?Thr?Pro?Pro?His?Leu?Thr?Ser

1155 1160

<210>54

<211>3597

<212>DNA

＜213〉rat

<220>

<221>CDS

<222>(40)..(3522)

<220>

＜223〉artificial sequence note: primer

<400>54

agctttacag?ctctctactt?ctcagtgcac?tgctcagtg?atg?gcc?ggt?gga?gtt 54

Met?Ala?Gly?Gly?Val

1 5

gtg?atc?ctc?ctg?tgt?ggc?tgg?gtc?ctg?gct?tcc?tgt?cat?ggg?tct?aac 102

Val?Ile?Leu?Leu?Cys?Gly?Trp?Val?Leu?Ala?Ser?Cys?His?Gly?Ser?Asn

10 15 20

ctg?gat?gtg?gag?gaa?ccc?atc?gtg?ttc?aga?gag?gat?gca?gcc?agc?ttt 150

Leu?Asp?Val?Glu?Glu?Pro?Ile?Val?Phe?Arg?Glu?Asp?Ala?Ala?Ser?Phe

25 30 35

gga?cag?act?gtg?gtg?cag?ttt?ggt?gga?tct?cga?ctc?gtg?gtg?gga?gcc 198

Gly?Gln?Thr?Val?Val?Gln?Phe?Gly?Gly?Ser?Arg?Leu?Val?Val?Gly?Ala

40 45 50

cct?ctg?gag?gcg?gtg?gca?gtc?aac?caa?aca?gga?cgg?ttg?tat?gac?tgt 246

Pro?Leu?Glu?Ala?Val?Ala?Val?Asn?Gln?Thr?Gly?Arg?Leu?Tyr?Asp?Cys

55 60 65

gca?cct?gcc?act?ggc?atg?tgc?cag?ccc?atc?gta?ctg?cgc?agt?ccc?cta 294

Ala?Pro?Ala?Thr?Gly?Met?Cys?Gln?Pro?Ile?Val?Leu?Arg?Ser?Pro?Leu

70 75 80 85

gag?gca?gtg?aac?atg?tcc?ctg?ggc?ctg?tct?ctg?gtg?act?gcc?acc?aat 342

Glu?Ala?Val?Asn?Met?Ser?Leu?Gly?Leu?Ser?Leu?Val?Thr?Ala?Thr?Asn

90 95 100

aac?gcc?cag?ttg?ctg?gct?tgt?ggt?cca?act?gca?cag?aga?gct?tgt?gtg 390

Asn?Ala?Gln?Leu?Leu?Ala?Cys?Gly?Pro?Thr?Ala?Gln?Arg?Ala?Cys?Val

105 110 115

aag?aac?atg?tat?gcg?aaa?ggt?tcc?tgc?ctc?ctt?ctc?ggc?tcc?agc?ttg 438

Lys?Asn?Met?Tyr?Ala?Lys?Gly?Ser?Cys?Leu?Leu?Leu?Gly?Ser?Ser?Leu

120 125 130

cag?ttc?atc?cag?gca?gtc?cct?gcc?tcc?atg?cca?gag?tgt?cca?aga?caa 486

Gln?Phe?Ile?Gln?Ala?Val?Pro?Ala?Ser?Met?Pro?Glu?Cys?Pro?Arg?Gln

135 140 145

gag?atg?gac?att?gct?ttc?ctg?att?gat?ggt?tct?ggc?agc?att?aac?caa 534

Glu?Met?Asp?Ile?Ala?Phe?Leu?Ile?Asp?Gly?Ser?Gly?Ser?Ile?Asn?Gln

150 155 160 165

agg?gac?ttt?gcc?cag?atg?aag?gac?ttt?gtc?aaa?gct?ttg?atg?gga?gag 582

Arg?Asp?Phe?Ala?Gln?Met?Lys?Asp?Phe?Val?Lys?Ala?Leu?Met?Gly?Glu

170 175 180

ttt?gcg?agc?acc?agc?acc?ttg?ttc?tcc?ctg?atg?caa?tac?tcg?aac?atc 630

Phe?Ala?Ser?Thr?Ser?Thr?Leu?Phe?Ser?Leu?Met?Gln?Tyr?Ser?Asn?Ile

185 190 195

ctg?aag?acc?cat?ttt?acc?ttc?act?gaa?ttc?aag?aac?atc?ctg?gac?cct 678

Leu?Lys?Thr?His?Phe?Thr?Phe?Thr?Glu?Phe?Lys?Asn?Ile?Leu?Asp?Pro

200 205 210

cag?agc?ctg?gtg?gat?ccc?att?gtc?cag?ctg?caa?ggc?ctg?acc?tac?aca 726

Gln?Ser?Leu?Val?Asp?Pro?Ile?Val?Gln?Leu?Gln?Gly?Leu?Thr?Tyr?Thr

215 220 225

gcc?aca?ggc?atc?cgg?aca?gtg?atg?gaa?gag?cta?ttt?cat?agc?aag?aat 774

Ala?Thr?Gly?Ile?Arg?Thr?Val?Met?Glu?Glu?Leu?Phe?His?Ser?Lys?Asn

230 235 240 245

ggg?tcc?cgt?aaa?agt?gcc?aag?aag?atc?ctc?ctt?gtc?atc?aca?gat?ggg 822

Gly?Ser?Arg?Lys?Ser?Ala?Lys?Lys?Ile?Leu?Leu?Val?Ile?Thr?Asp?Gly

250 255 260

cag?aaa?tac?aga?gac?ccc?ctg?gag?tat?agt?gat?gtc?att?ccc?gcc?gca 870

Gln?Lys?Tyr?Arg?Asp?Pro?Leu?Glu?Tyr?Ser?Asp?Val?Ile?Pro?Ala?Ala

265 270 275

gac?aaa?gct?ggc?atc?att?cgt?tat?gct?att?ggg?gtg?gga?gat?gcc?ttc 918

Asp?Lys?Ala?Gly?Ile?Ile?Arg?Tyr?Ala?Ile?Gly?Val?Gly?Asp?Ala?Phe

280 285 290

cag?gag?ccc?act?gcc?ctg?aag?gag?ctg?aac?acc?att?ggc?tca?gct?ccc 966

Gln?Glu?Pro?Thr?Ala?Leu?Lys?Glu?Leu?Asn?Thr?Ile?Gly?Ser?Ala?Pro

295 300 305

cca?cag?gac?cac?gtg?ttc?aag?gta?ggc?aac?ttt?gca?gca?ctt?cgc?agc 1014

Pro?Gln?Asp?His?Val?Phe?Lys?Val?Gly?Asn?Phe?Ala?Ala?Leu?Arg?Ser

310 315 320 325

atc?cag?agg?caa?ctt?cag?gag?aaa?atc?ttc?gcc?att?gag?gga?act?caa 1062

Ile?Gln?Arg?Gln?Leu?Gln?Glu?Lys?Ile?Phe?Ala?Ile?Glu?Gly?Thr?Gln

330 335 340

tca?agg?tca?agt?agt?tcc?ttt?cag?cac?gag?atg?tca?caa?gaa?ggt?ttc 1110

Ser?Arg?Ser?Ser?Ser?Ser?Phe?Gln?His?Glu?Met?Ser?Gln?Glu?Gly?Phe

345 350 355

agt?tca?gct?ctc?aca?tcg?gat?gga?ccc?gtt?ctg?ggg?gcc?gtg?gga?agc 1158

Ser?Ser?Ala?Leu?Thr?Ser?Asp?Gly?Pro?Val?Leu?Gly?Ala?Val?Gly?Ser

360 365 370

ttc?agc?tgg?tcc?gga?ggt?gcc?ttc?tta?tat?ccc?cca?aat?acg?aga?ccc 1206

Phe?Ser?Trp?Ser?Gly?Gly?Ala?Phe?Leu?Tyr?Pro?Pro?Asn?Thr?Arg?Pro

375 380 385

acc?ttt?atc?aac?atg?tct?cag?gag?aat?gtg?gac?atg?aga?gac?tcc?tac 1254

Thr?Phe?Ile?Asn?Met?Ser?Gln?Glu?Asn?Val?Asp?Met?Arg?Asp?Ser?Tyr

390 395 400 405

ctg?ggt?tac?tcc?acc?gca?gtg?gcc?ttt?tgg?aag?ggg?gtt?cac?agc?ctg 1302

Leu?Gly?Tyr?Ser?Thr?Ala?Val?Ala?Phe?Trp?Lys?Gly?Val?His?Ser?Leu

410 415 420

atc?ctg?ggg?gcc?ccg?cgt?cac?cag?cac?acg?ggg?aag?gtt?gtc?atc?ttt 1350

Ile?Leu?Gly?Ala?Pro?Arg?His?Gln?His?Thr?Gly?Lys?Val?Val?Ile?Phe

425 430 435

acc?cag?gaa?gcc?agg?cat?tgg?agg?ccc?aag?tct?gaa?gtc?aga?ggg?aca 1398

Thr?Gln?Glu?Ala?Arg?His?Trp?Arg?Pro?Lys?Ser?Glu?Val?Arg?Gly?Thr

440 445 450

cag?atc?ggc?tcc?tac?ttc?ggg?gcc?tct?ctc?tgt?tct?gtg?gac?gtg?gat 1446

Gln?Ile?Gly?Ser?Tyr?Phe?Gly?Ala?Ser?Leu?Cys?Ser?Val?Asp?Val?Asp

455 460 465

aga?gat?ggc?agc?acy?gac?ctg?gtc?ctg?atc?gga?gcc?ccc?cat?tac?tat 1494

Arg?Asp?Gly?Ser?Xaa?Asp?Leu?Val?Leu?Ile?Gly?Ala?Pro?His?Tyr?Tyr

470 475 480 485

gag?cag?acc?cga?ggg?ggg?cag?gtc?tca?gtg?ttc?ccc?gtg?ccc?ggt?gtg 1542

Glu?Gln?Thr?Arg?Gly?Gly?Gln?Val?Ser?Val?Phe?Pro?Val?Pro?Gly?Val

490 495 500

agg?ggc?agg?tgg?cag?tgt?gag?gcc?acc?ctc?cac?ggg?gag?cag?ggc?cat 1590

Arg?Gly?Arg?Trp?Gln?Cys?Glu?Ala?Thr?Leu?His?Gly?Glu?Gln?Gly?His

505 510 515

cct?tgg?ggc?cgc?ttt?ggg?gtg?gct?ctg?aca?gtg?ctg?ggg?gac?gta?aac 1638

Pro?Trp?Gly?Arg?Phe?Gly?Val?Ala?Leu?Thr?Val?Leu?Gly?Asp?Val?Asn

520 525 530

ggg?gac?aat?ctg?gca?gac?gtg?gct?att?ggt?gcc?cct?gga?gag?gag?gag 1686

Gly?Asp?Asn?Leu?Ala?Asp?Val?Ala?Ile?Gly?Ala?Pro?Gly?Glu?Glu?Glu

535 540 545

agc?aga?ggt?gct?gtc?tac?ata?ttt?cat?gga?gcc?tcg?aga?ctg?gag?atc 1734

Ser?Arg?Gly?Ala?Val?Tyr?Ile?Phe?His?Gly?Ala?Ser?Arg?Leu?Glu?Ile

550 555 560 565

atg?ccc?tca?ccc?agc?cag?cgg?gtc?act?ggc?tcc?cag?ctc?tcc?ctg?aga 1782

Met?Pro?Ser?Pro?Ser?Gln?Arg?Val?Thr?Gly?Ser?Gln?Leu?Ser?Leu?Arg

570 575 580

ctg?cag?tat?ttt?ggg?cag?tca?ttg?agt?ggg?ggt?cag?gac?ctt?aca?cag 1830

Leu?Gln?Tyr?Phe?Gly?Gln?Ser?Leu?Ser?Gly?Gly?Gln?Asp?Leu?Thr?Gln

585 590 595

gat?ggc?ctg?gtg?gac?ctg?gcc?gtg?gga?gcc?cag?ggg?cac?gta?ctg?ctg 1878

Asp?Gly?Leu?Val?Asp?Leu?Ala?Val?Gly?Ala?Gln?Gly?His?Val?Leu?Leu

600 605 610

ctc?agg?agt?ctg?cct?ctg?ctg?aaa?gtg?gag?ctc?tcc?ata?aga?ttc?gcc 1926

Leu?Arg?Ser?Leu?Pro?Leu?Leu?Lys?Val?Glu?Leu?Ser?Ile?Arg?Phe?Ala

615 620 625

ccc?atg?gag?gtg?gca?aag?gct?gtg?tac?cag?tgc?tgg?gaa?agg?act?ccc 1974

Pro?Met?Glu?Val?Ala?Lys?Ala?Val?Tyr?Gln?Cys?Trp?GLu?Arg?Thr?Pro

630 635 640 645

act?gtc?ctc?gaa?gct?gga?gag?gcc?act?gtc?tgt?ctc?act?gtc?cac?aaa 2022

Thr?Val?Leu?Glu?Ala?Gly?Glu?Ala?Thr?Val?Cys?Leu?Thr?Val?His?Lys

650 655 660

ggc?tca?cct?gac?ctg?tta?ggt?aat?gtc?caa?ggc?tct?gtc?agg?tat?gat 2070

Gly?Ser?Pro?Asp?Leu?Leu?Gly?Asn?Val?Gln?Gly?Ser?Val?Arg?Tyr?Asp

665 670 675

ctg?gcg?tta?gat?ccg?ggc?cgc?ctg?att?tct?cgt?gcc?att?ttt?gat?gag 2118

Leu?Ala?Leu?Asp?Pro?Gly?Arg?Leu?Ile?Ser?Arg?Ala?Ile?Phe?Asp?Glu

680 685 690

act?aag?aac?tgc?act?ttg?acg?gga?agg?aag?act?ctg?ggg?ctt?ggt?gat 2166

Thr?Lys?Asn?Cys?Thr?Leu?Thr?Gly?Arg?Lys?Thr?Leu?Gly?Leu?Gly?Asp

695 700 705

cac?tgc?gaa?aca?gtg?aag?ctg?ctt?ttg?ccg?gac?tgt?gtg?gaa?gat?gca 2214

His?Cys?Glu?Thr?Val?Lys?Leu?Leu?Leu?Pro?Asp?Cys?Val?Glu?Asp?Ala

710 715 720 725

gtg?agc?cct?atc?atc?ctg?cgc?ctc?aac?ttt?tcc?ctg?gtg?aga?gac?tct 2262

Val?Ser?Pro?Ile?Ile?Leu?Arg?Leu?Asn?Phe?Ser?Leu?Val?Arg?Asp?Ser

730 735 740

gct?tca?ccc?agg?aac?ctg?cat?cct?gtg?ctg?gct?gtg?ggc?tca?caa?gac 2310

Ala?Ser?Pro?Arg?Asn?Leu?His?Pro?Val?Leu?Ala?Val?Gly?Ser?Gln?Asp

745 750 755

cac?ata?act?gct?tct?ctg?ccg?ttt?gag?aag?aac?tgt?aag?caa?gaa?ctc 2358

His?Ile?Thr?Ala?Ser?Leu?Pro?Phe?Glu?Lys?Asn?Cys?Lys?Gln?Glu?Leu

760 765 770

ctg?tgt?gag?ggg?gac?ctg?ggc?atc?agc?ttt?aac?ttc?tca?ggc?ctg?cag 2406

Leu?Cys?Glu?Gly?Asp?Leu?Gly?Ile?Ser?Phe?Asn?Phe?Ser?Gly?Leu?Gln

775 780 785

gtc?ttg?gtg?gtg?gga?ggc?tcc?cca?gag?ctc?act?gtg?aca?gtc?act?gtg 2454

Val?Leu?Val?Val?Gly?Gly?Ser?Pro?Glu?Leu?Thr?Val?Thr?Val?Thr?Val

790 795 800 805

tgg?aat?gag?ggt?gag?gac?agc?tat?gga?act?tta?gtc?aag?ttc?tac?tac 2502

Trp?Asn?Glu?Gly?Glu?Asp?Ser?Tyr?Gly?Thr?Leu?Val?Lys?Phe?Tyr?Tyr

810 815 820

cca?gca?ggg?cta?tct?tac?cga?cgg?gta?aca?ggg?act?cag?caa?cct?cat 2550

Pro?Ala?Gly?Leu?Ser?Tyr?Arg?Arg?Val?Thr?Gly?Thr?Gln?Gln?Pro?His

825 830 835

cag?tac?cca?cta?cgc?ttg?gcc?tgt?gag?gct?gag?ccc?gct?gcc?cag?gag 2598

Gln?Tyr?Pro?Leu?Arg?Leu?Ala?Cys?Glu?Ala?Glu?Pro?Ala?Ala?Gln?Glu

840 845 850

gac?ctg?agg?agc?agc?agc?tgt?agc?att?aat?cac?ccc?atc?ttc?cga?gaa 2646

Asp?Leu?Arg?Ser?Ser?Ser?Cys?Ser?Ile?Asn?His?Pro?Ile?Phe?Arg?Glu

855 860 865

ggt?gca?aag?acc?acc?ttc?atg?atc?aca?ttc?gat?gtc?tcc?tac?aag?gcc 2694

Gly?Ala?Lys?Thr?Thr?Phe?Met?Ile?Thr?Phe?Asp?Val?Ser?Tyr?Lys?Ala

870 875 880 885

ttc?cta?gga?gac?agg?ttg?ctt?ctg?agg?gcc?aaa?gcc?agc?agt?gag?aat 2742

Phe?Leu?Gly?Asp?Arg?Leu?Leu?Leu?Arg?Ala?Lys?Ala?Ser?Ser?Glu?Asn

890 895 900

aat?aag?cct?gat?acc?aac?aag?act?gcc?ttc?cag?ctg?gag?ctc?cca?gtg 2790

Asn?Lys?Pro?Asp?Thr?Asn?Lys?Thr?Ala?Phe?Gln?Leu?Glu?Leu?Pro?Val

905 910 915

aag?tac?acc?gtc?tat?acc?ctg?atc?agt?agg?caa?gaa?gat?tcc?acc?aac 2838

Lys?Tyr?Thr?Val?Tyr?Thr?Leu?Ile?Ser?Arg?Gln?Glu?Asp?Ser?Thr?Asn

920 925 930

cat?gtc?aac?ttt?tca?tct?tcc?cac?ggg?ggg?aga?agg?caa?gaa?gcc?gca 2886

His?Val?Asn?Phe?Ser?Ser?Ser?His?Gly?Gly?Arg?Arg?Gln?Glu?Ala?Ala

935 940 945

cat?cgc?tat?cgt?gtg?aat?aac?ctg?agt?cca?ctg?aag?ctg?gcc?gtc?aga 2934

His?Arg?Tyr?Arg?Val?Asn?Asn?Leu?Ser?Pro?Leu?Lys?Leu?Ala?Val?Arg

950 955 960 965

gtt?aac?ttc?tgg?gtc?cct?gtc?ctt?ctg?aac?ggt?gtg?gct?gtg?tgg?gac 2982

Val?Asn?Phe?Trp?Val?Pro?Val?Leu?Leu?Asn?Gly?Val?Ala?Val?Trp?Asp

970 975 980

gtg?act?ctg?agc?agc?cca?gca?cag?ggt?gtc?tcc?tgc?gtg?tcc?cag?atg 3030

Val?Thr?Leu?Ser?Ser?Pro?Ala?Gln?Gly?Val?Ser?Cys?Val?Ser?Gln?Met

985 990 995

aaa?cct?cct?cag?aat?ccc?gac?ttt?ctg?acc?cag?att?cag?aga?cgt?tct 3078

Lys?Pro?Pro?Gln?Asn?Pro?Asp?Phe?Leu?Thr?Gln?Ile?Gln?Arg?Arg?Ser

1000 1005 1010

gtg?ctg?gac?tgc?tcc?att?gct?gac?tgc?ctg?cac?ttc?cgc?tgt?gac?atc 3126

Val?Leu?Asp?Cys?Ser?Ile?Ala?Asp?Cys?Leu?His?Phe?Arg?Cys?Asp?Ile

1015 1020 1025

ccc?tcc?ttg?gac?atc?cag?gat?gaa?ctt?gac?ttc?att?ctg?agg?ggc?aac 3174

Pro?Ser?Leu?Asp?Ile?Gln?Asp?Glu?Leu?Asp?Phe?Ile?Leu?Arg?Gly?Asn

1030 1035 1040 1045

ctc?agc?ttc?ggc?tgg?gtc?agt?cag?aca?ttg?cag?gaa?aag?gtg?ttg?ctt 3222

Leu?Ser?Phe?Gly?Trp?Val?Ser?Gln?Thr?Leu?Gln?Glu?Lys?Val?Leu?Leu

1050 1055 1060

gtg?agt?gag?gct?gaa?atc?act?ttc?gac?aca?tct?gtg?tac?tcc?cag?ctg 3270

Val?Ser?Glu?Ala?Glu?Ile?Thr?Phe?Asp?Thr?Ser?Val?Tyr?Ser?Gln?Leu

1065 1070 1075

cca?gga?cag?gag?gca?ttt?ctg?aga?gcc?cag?gtg?gag?aca?acg?tta?gaa 3318

Pro?Gly?Gln?Glu?Ala?Phe?Leu?Arg?Ala?Gln?Val?Glu?Thr?Thr?Leu?Glu

1080 1085 1090

gaa?tac?gtg?gtc?tat?gag?ccc?atc?ttc?ctc?gtg?gcg?ggc?agc?tcg?gtg 3366

Glu?Tyr?Val?Val?Tyr?Glu?Pro?Ile?Phe?Leu?Val?Ala?Gly?Ser?Ser?Val

1095 1100 1105

gga?ggt?ctg?ctg?tta?ctg?gct?ctc?atc?aca?gtg?gta?ctg?tac?aag?ctt 3414

Gly?Gly?Leu?Leu?Leu?Leu?Ala?Leu?Ile?Thr?Val?Val?Leu?Tyr?Lys?Leu

1110 1115 1120 1125

ggc?ttc?tyc?aaa?cgt?cag?tac?aaa?gaa?atg?ctg?gac?ggc?aag?gct?gca 3462

Gly?Phe?Xaa?Lys?Arg?Gln?Tyr?Lys?Glu?Met?Leu?Asp?Gly?Lys?Ala?Ala

1130 1135 1140

gat?cct?gtc?aca?gcc?ggc?cag?gca?gat?ttc?ggc?tgt?gag?act?cct?cca 3510

Asp?Pro?Val?Thr?Ala?Gly?Gln?Ala?Asp?Phe?Gly?Cys?Glu?Thr?Pro?Pro

1145 1150 1155

tat?ctc?gtg?agc?taggaatcca?ctctcctgcc?tatctctgca?atgaagattg 3562

Tyr?Leu?Val?Ser

1160

gtcctgccta?tgagtctact?ggcatgggaa?cgagt 3597

<210>55

<211>1161

<212>PRT

＜213〉rat

<400>55

Met?Ala?Gly?Gly?Val?Val?Ile?Leu?Leu?Cys?Gly?Trp?Val?Leu?Ala?Ser

1 5 10 15

Cys?His?Gly?Ser?Asn?Leu?Asp?Val?Glu?Glu?Pro?Ile?Val?Phe?Arg?Glu

20 25 30

Asp?Ala?Ala?Ser?Phe?Gly?Gln?Thr?Val?Val?Gln?Phe?Gly?Gly?Ser?Arg

35 40 45

Leu?Val?Val?Gly?Ala?Pro?Leu?Glu?Ala?Val?Ala?Val?Asn?Gln?Thr?Gly

50 55 60

Arg?Leu?Tyr?Asp?Cys?Ala?Pro?Ala?Thr?Gly?Met?Cys?Gln?Pro?Ile?Val

65 70 75 80

Leu?Arg?Ser?Pro?Leu?Glu?Ala?Val?Asn?Met?Ser?Leu?Gly?Leu?Ser?Leu

85 90 95

Val?Thr?Ala?Thr?Asn?Asn?Ala?Gln?Leu?Leu?Ala?Cys?Gly?Pro?Thr?Ala

100 105 110

Gln?Arg?Ala?Cys?Val?Lys?Asn?Met?Tyr?Ala?Lys?Gly?Ser?Cys?Leu?Leu

115 120 125

Leu?Gly?Ser?Ser?Leu?Gln?Phe?Ile?Gln?Ala?Val?Pro?Ala?Ser?Met?Pro

130 135 140

Glu?Cys?Pro?Arg?Gln?Glu?Met?Asp?Ile?Ala?Phe?Leu?Ile?Asp?Gly?Ser

145 150 155 160

Gly?Ser?Ile?Asn?Gln?Arg?Asp?Phe?Ala?Gln?Met?Lys?Asp?Phe?Val?Lys

165 170 175

Ala?Leu?Met?Gly?Glu?Phe?Ala?Ser?Thr?Ser?Thr?Leu?Phe?Ser?Leu?Met

180 185 190

Gln?Tyr?Ser?Asn?Ile?Leu?Lys?Thr?His?Phe?Thr?Phe?Thr?Glu?Phe?Lys

195 200 205

Asn?Ile?Leu?Asp?Pro?Gln?Ser?Leu?Val?Asp?Pro?Ile?Val?Gln?Leu?Gln

210 215 220

Gly?Leu?Thr?Tyr?Thr?Ala?Thr?Gly?Ile?Arg?Thr?Val?Met?Glu?Glu?Leu

225 230 235 240

Phe?His?Ser?Lys?Asn?Gly?Ser?Arg?Lys?Ser?Ala?Lys?Lys?Ile?Leu?Leu

245 250 255

Val?Ile?Thr?Asp?Gly?Gln?Lys?Tyr?Arg?Asp?Pro?Leu?Glu?Tyr?Ser?Asp

260 265 270

Val?Ile?Pro?Ala?Ala?Asp?Lys?Ala?Gly?Ile?Ile?Arg?Tyr?Ala?Ile?Gly

275 280 285

Val?Gly?Asp?Ala?Phe?Gln?Glu?Pro?Thr?Ala?Leu?Lys?Glu?Leu?Asn?Thr

290 295 300

Ile?Gly?Ser?Ala?Pro?Pro?Gln?Asp?His?Val?Phe?Lys?Val?Gly?Asn?Phe

305 310 315 320

Ala?Ala?Leu?Arg?Ser?Ile?Gln?Arg?Gln?Leu?Gln?Glu?Lys?Ile?Phe?Ala

325 330 335

Ile?Glu?Gly?Thr?Gln?Ser?Arg?Ser?Ser?Ser?Ser?Phe?Gln?His?Glu?Met

340 345 350

Ser?Gln?Glu?Gly?Phe?Ser?Ser?Ala?Leu?Thr?Ser?Asp?Gly?Pro?Val?Leu

355 360 365

Gly?Ala?Val?Gly?Ser?Phe?Ser?Trp?Ser?Gly?Gly?Ala?Phe?Leu?Tyr?Pro

370 375 380

Pro?Asn?Thr?Arg?Pro?Thr?Phe?Ile?Asn?Met?Ser?Gln?Glu?Asn?Val?Asp

385 390 395 400

Met?Arg?Asp?Ser?Tyr?Leu?Gly?Tyr?Ser?Thr?Ala?Val?Ala?Phe?Trp?Lys

405 410 415

Gly?Val?His?Ser?Leu?Ile?Leu?Gly?Ala?Pro?Arg?His?Gln?His?Thr?Gly

420 425 430

Lys?Val?Val?Ile?Phe?Thr?Gln?Glu?Ala?Arg?His?Trp?Arg?Pro?Lys?Ser

435 440 445

Glu?Val?Arg?Gly?Thr?Gln?Ile?Gly?Ser?Tyr?Phe?Gly?Ala?Ser?Leu?Cys

450 455 460

Ser?Val?Asp?Val?Asp?Arg?Asp?Gly?Ser?Xaa?Asp?Leu?Val?Leu?Ile?Gly

465 470 475 480

Ala?Pro?His?Tyr?Tyr?Glu?Gln?Thr?Arg?Gly?Gly?Gln?Val?Ser?Val?Phe

485 490 495

Pro?Val?Pro?Gly?Val?Arg?Gly?Arg?Trp?Gln?Cys?Glu?Ala?Thr?Leu?His

500 505 510

Gly?Glu?Gln?Gly?His?Pro?Trp?Gly?Arg?Phe?Gly?Val?Ala?Leu?Thr?Val

515 520 525

Leu?Gly?Asp?Val?Asn?Gly?Asp?Asn?Leu?Ala?Asp?Val?Ala?Ile?Gly?Ala

530 535 540

Pro?Gly?Glu?Glu?Glu?Ser?Arg?Gly?Ala?Val?Tyr?Ile?Phe?His?Gly?Ala

545 550 555 560

Ser?Arg?Leu?Glu?Ile?Met?Pro?Ser?Pro?Ser?Gln?Arg?Val?Thr?Gly?Ser

565 570 575

Gln?Leu?Ser?Leu?Arg?Leu?Gln?Tyr?Phe?Gly?Gln?Ser?Leu?Ser?Gly?Gly

580 585 590

Gln?Asp?Leu?Thr?Gln?Asp?Gly?Leu?Val?Asp?Leu?Ala?Val?Gly?Ala?Gln

595 600 605

Gly?His?Val?Leu?Leu?Leu?Arg?Ser?Leu?Pro?Leu?Leu?Lys?Val?Glu?Leu

610 615 620

Ser?Ile?Arg?Phe?Ala?Pro?Met?Glu?Val?Ala?Lys?Ala?Val?Tyr?Gln?Cys

625 630 635 640

Trp?Glu?Arg?Thr?Pro?Thr?Val?Leu?Glu?Ala?Gly?Glu?Ala?Thr?Val?Cys

645 650 655

Leu?Thr?Val?His?Lys?Gly?Ser?Pro?Asp?Leu?Leu?Gly?Asn?Val?Gln?Gly

660 665 670

Ser?Val?Arg?Tyr?Asp?Leu?Ala?Leu?Asp?Pro?Gly?Arg?Leu?Ile?Ser?Arg

675 680 685

Ala?Ile?Phe?Asp?Glu?Thr?Lys?Asn?Cys?Thr?Leu?Thr?Gly?Arg?Lys?Thr

690 695 700

Leu?Gly?Leu?Gly?Asp?His?Cys?Glu?Thr?Val?Lys?Leu?Leu?Leu?Pro?Asp

705 710 715 720

Cys?Val?Glu?Asp?Ala?Val?Ser?Pro?Ile?Ile?Leu?Arg?Leu?Asn?Phe?Ser

725 730 735

Leu?Val?Arg?Asp?Ser?Ala?Ser?Pro?Arg?Asn?Leu?His?Pro?Val?Leu?Ala

740 745 750

Val?Gly?Ser?Gln?Asp?His?Ile?Thr?Ala?Ser?Leu?Pro?Phe?Glu?Lys?Asn

755 760 765

Cys?Lys?Gln?Glu?Leu?Leu?Cys?Glu?Gly?Asp?Leu?Gly?Ile?Ser?Phe?Asn

770 775 780

Phe?Ser?Gly?Leu?Gln?Val?Leu?Val?Val?Gly?Gly?Ser?Pro?Glu?Leu?Thr

785 790 795 800

Val?Thr?Val?Thr?Val?Trp?Asn?Glu?Gly?Glu?Asp?Ser?Tyr?Gly?Thr?Leu

805 810 815

Val?Lys?Phe?Tyr?Tyr?Pro?Ala?Gly?Leu?Ser?Tyr?Arg?Arg?Val?Thr?Gly

820 825 830

Thr?Gln?Gln?Pro?His?Gln?Tyr?Pro?Leu?Arg?Leu?Ala?Cys?Glu?Ala?Glu

835 840 845

Pro?Ala?Ala?Gln?Glu?Asp?Leu?Arg?Ser?Ser?Ser?Cys?Ser?Ile?Asn?His

850 855 860

Pro?Ile?Phe?Arg?Glu?Gly?Ala?Lys?Thr?Thr?Phe?Met?Ile?Thr?Phe?Asp

865 870 875 880

Val?Ser?Tyr?Lys?Ala?Phe?Leu?Gly?Asp?Arg?Leu?Leu?Leu?Arg?Ala?Lys

885 890 895

Ala?Ser?Ser?Glu?Asn?Asn?Lys?Pro?Asp?Thr?Asn?Lys?Thr?Ala?Phe?Gln

900 905 910

Leu?Glu?Leu?Pro?Val?Lys?Tyr?Thr?Val?Tyr?Thr?Leu?Ile?Ser?Arg?Gln

915 920 925

Glu?Asp?Ser?Thr?Asn?His?Val?Asn?Phe?Ser?Ser?Ser?His?Gly?Gly?Arg

930 935 940

Arg?Gln?Glu?Ala?Ala?His?Arg?Tyr?Arg?Val?Asn?Asn?Leu?Ser?Pro?Leu

945 950 955 960

Lys?Leu?Ala?Val?Arg?Val?Asn?Phe?Trp?Val?Pro?Val?Leu?Leu?Asn?Gly

965 970 975

Val?Ala?Val?Trp?Asp?Val?Thr?Leu?Ser?Ser?Pro?Ala?Gln?Gly?Val?Ser

980 985 990

Cys?Val?Ser?Gln?Met?Lys?Pro?Pro?Gln?Asn?Pro?Asp?Phe?Leu?Thr?Gln

995 1000 1005

Ile?Gln?Arg?Arg?Ser?Val?Leu?Asp?Cys?Ser?Ile?Ala?Asp?Cys?Leu?His

1010 1015 1020

Phe?Arg?Cys?Asp?Ile?Pro?Ser?Leu?Asp?Ile?Gln?Asp?Glu?Leu?Asp?Phe

1025 1030 1035 1040

Ile?Leu?Arg?Gly?Asn?Leu?Ser?Phe?Gly?Trp?Val?Ser?Gln?Thr?Leu?Gln

1045 1050 1055

Glu?Lys?Val?Leu?Leu?Val?Ser?Glu?Ala?Glu?Ile?Thr?Phe?Asp?Thr?Ser

1060 1065 1070

Val?Tyr?Ser?Gln?Leu?Pro?Gly?Gln?Glu?Ala?Phe?Leu?Arg?Ala?Gln?Val

1075 1080 1085

Glu?Thr?Thr?Leu?Glu?Glu?Tyr?Val?Val?Tyr?Glu?Pro?Ile?Phe?Leu?Val

1090 1095 1100

Ala?Gly?Ser?Ser?Val?Gly?Gly?Leu?Leu?Leu?Leu?Ala?Leu?Ile?Thr?Val

1105 1110 1115 1120

Val?Leu?Tyr?Lys?Leu?Gly?Phe?Xaa?Lys?Arg?Gln?Tyr?Lys?Glu?Met?Leu

1125 1130 1135

Asp?Gly?Lys?Ala?Ala?Asp?Pro?Val?Thr?Ala?Gly?Gln?Ala?Asp?Phe?Gly

1140 1145 1150

Cys?Glu?Thr?Pro?Pro?Tyr?Leu?Val?Ser

1155 1160

<210>56

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>56

cctgtcatgg?gtctaacctg 20

<210>57

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>57

aggttagacc?catgacagg 19

<210>58

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>58

ggccttgcag?ctggacaatg 20

<210>59

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>59

ccaaagctgg?ctgcatcctc?tc 22

<210>60

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>60

ccgcctgcca?ctggcgtgtg?c 21

<210>61

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>61

cccagatgaa?ggacttcgtc?aa 22

<210>62

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>62

gctgggatca?ttcgctatgc 20

<210>63

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>63

caatggatgg?accagttctg?g 21

<210>64

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>64

cagatcggct?cctactttgg 20

<210>65

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>65

catggagcct?cgagacagg 19

<210>66

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>66

ccactgtcct?cgaagctgga?g 21

<210>67

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>67

cttcgtcctg?tgctggctgt?gggctc 26

<210>68

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>68

cgcctggcat?gtgaggctga?g 21

<210>69

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>69

ccgtgatcag?taggcaggaa?g 21

<210>70

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>70

gtcacagagg?gaacctcc 18

<210>71

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>71

gctcctgagt?gaggctgaaa?tca 23

<210>72

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>72

gagatgctgg?atctaccatc?tgc 23

<210>73

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>73

ctgagctggg?agatttttat?gg 22

<210>74

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>74

gtggatcagc?actgaaatct?g 21

<210>75

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>75

cgtttgaaga?agccaagctt?g 21

<210>76

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>76

cacagcggag?gtgcaggcag 20

<210>77

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>77

ctcactgctt?gcgctggc 18

<210>78

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>78

cggtaagata?gctctgctgg 20

<210>79

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>79

gagcccacag?ccagcacagg 20

<210>80

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>80

gatccaacgc?cagatcatac?c 21

<210>81

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>81

cacggccagg?tccaccaggc 20

<210>82

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>82

cacgtcccct?agcactgtca?g 21

<210>83

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>83

ttgacgaagt?ccttcatctg?gg 22

<210>84

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>84

gaactgcaag?ctggagccca?g 21

<210>85

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>85

ctggatgctg?cgaagtgcta?c 21

<210>86

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>86

gccttggagc?tggacgatgg?c 21

<210>87

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>87

gtaagatctc?cagagtgtcc?aagacaagag?atg 33

<210>88

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>88

cttctcgagt?gtgagagctg?aactgaaacc?ttc 33

<210>89

<211>32

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>89

cgctgtgacg?tcagagttga?gtccaaatat?gg 32

<210>90

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>90

ggtgacacta?tagaataggg?c 21

<210>91

<211>18

<212>DNA

＜213〉mice

<400>91

aagcaggagc?tcctgtgt 18

<210>92

<211>852

<212>DNA

＜213〉rabbit

<220>

<221>CDS

<222>(61)..(852)

<400>92

tgatctccct?ccaggccact?gttccctctc?cacttcccct?caccgctgca?ctgctcagag?60

atg?gcc?ctt?ggg?gct?gtg?gtc?ctc?ctt?ggg?gtc?ctg?gct?tct?tac?cac 108

Met?Ala?Leu?Gly?Ala?Val?Val?Leu?Leu?Gly?Val?Leu?Ala?Ser?Tyr?His

1 5 10 15

gga?ttc?aac?ttg?gac?gtg?atg?agc?ggt?gat?ctt?cca?gga?aga?cgc?agc 156

Gly?Phe?Asn?Leu?Asp?Val?Met?Ser?Gly?Asp?Leu?Pro?Gly?Arg?Arg?Ser

20 25 30

ggg?ctt?cgg?gca?gag?cgt?gat?gca?gtt?tgg?gga?tct?cga?ctc?gtg?gtg 204

Gly?Leu?Arg?Ala?Glu?Arg?Asp?Ala?Val?Trp?Gly?Ser?Arg?Leu?Val?Val

35 40 45

gga?gcc?ccc?ctg?gcg?gtg?gtg?tcg?gcc?aac?cac?aca?gga?cgg?ctg?tac 252

Gly?Ala?Pro?Leu?Ala?Val?Val?Ser?Ala?Asn?His?Thr?Gly?Arg?Leu?Tyr

50 55 60

gag?tgt?gcg?cct?gcc?tcc?ggc?acc?tgc?acg?ccc?att?ttc?cca?ttc?atg 300

Glu?Cys?Ala?Pro?Ala?Ser?Gly?Thr?Cys?Thr?Pro?Ile?Phe?Pro?Phe?Met

65 70 75 80

ccc?ccc?gaa?gcc?gtg?aac?atg?tcc?ctg?ggc?ctg?tcc?ctg?gca?gcc?tcc 348

Pro?Pro?Glu?Ala?Val?Asn?Met?Ser?Leu?Gly?Leu?Ser?Leu?Ala?Ala?Ser

85 90 95

ccc?aac?cat?tcc?cag?ctg?ctg?gct?tgt?ggc?ccg?acc?gtg?cat?aga?gcc 396

Pro?Asn?His?Ser?Gln?Leu?Leu?Ala?Cys?Gly?Pro?Thr?Val?His?Arg?Ala

100 105 110

tgc?ggg?gag?gac?gtg?tac?gcc?cag?ggt?ttc?tgt?gtg?ctg?ctg?gat?gcc 444

Cys?Gly?Glu?Asp?Val?Tyr?Ala?Gln?Gly?Phe?Cys?Val?Leu?Leu?Asp?Ala

115 120 125

cac?gca?cag?ccc?atc?ggg?act?gtg?cca?gct?gcc?ctg?ccc?gag?tgc?cca 492

His?Ala?Gln?Pro?Ile?Gly?Thr?Val?Pro?Ala?Ala?Leu?Pro?Glu?Cys?Pro

130 135 140

gat?caa?gag?atg?gac?att?gtc?ttc?ctg?att?gac?ggc?tct?ggc?agc?att 540

Asp?Gln?Glu?Met?Asp?Ile?Val?Phe?Leu?Ile?Asp?Gly?Ser?Gly?Ser?Ile

145 150 155 160

agc?tca?aat?gac?ttc?cgc?aag?atg?aag?gac?ttt?gtc?aga?gct?gtg?atg 588

Ser?Ser?Asn?Asp?Phe?Arg?Lys?Met?Lys?Asp?Phe?Val?Arg?Ala?Val?Met

165 170 175

gac?cag?ttc?aag?gac?acc?aac?acc?cag?ttc?tcg?ctg?atg?cag?tac?tcc 636

Asp?Gln?Phe?Lys?Asp?Thr?Asn?Thr?Gln?Phe?Ser?Leu?Met?Gln?Tyr?Ser

180 185 190

aat?gtg?ctg?gtg?aca?cat?ttc?acc?ttc?agc?agc?ttc?cgg?aac?agc?tcc 684

Asn?Val?Leu?Val?Thr?His?Phe?Thr?Phe?Ser?Ser?Phe?Arg?Asn?Ser?Ser

195 200 205

aat?cct?cag?ggc?cta?gtg?gag?ccc?att?gtg?cag?ctg?aca?ggc?ctc?acg 732

Asn?Pro?Gln?Gly?Leu?Val?Glu?Pro?Ile?Val?Gln?Leu?Thr?Gly?Leu?Thr

210 215 220

ttc?acg?gcc?aca?ggg?atc?ctg?aaa?gtg?gtg?aca?gag?ctg?ttt?caa?acc 780

Phe?Thr?Ala?Thr?Gly?Ile?Leu?Lys?Val?Val?Thr?Glu?Leu?Phe?Gln?Thr

225 230 235 240

aag?aac?ggg?gcc?cgc?gaa?agt?gcc?aag?aag?atc?ctc?atc?gtc?atc?aca 828

Lys?Asn?Gly?Ala?Arg?Glu?Ser?Ala?Lys?Lys?Ile?Leu?Ile?Val?Ile?Thr

245 250 255

gat?ggg?cag?aag?tac?aaa?gcg?gca 852

Asp?Gly?Gln?Lys?Tyr?Lys?Ala?Ala

260

<210>93

<211>264

<212>PRT

＜213〉rabbit

<400>93

Met?Ala?Leu?Gly?Ala?Val?Val?Leu?Leu?Gly?Val?Leu?Ala?Ser?Tyr?His

1 5 10 15

Gly?Phe?Asn?Leu?Asp?Val?Met?Ser?Gly?Asp?Leu?Pro?Gly?Arg?Arg?Ser

20 25 30

Gly?Leu?Arg?Ala?Glu?Arg?Asp?Ala?Val?Trp?Gly?Ser?Arg?Leu?Val?Val

35 40 45

Gly?Ala?Pro?Leu?Ala?Val?Val?Ser?Ala?Asn?His?Thr?Gly?Arg?Leu?Tyr

50 55 60

Glu?Cys?Ala?Pro?Ala?Ser?Gly?Thr?Cys?Thr?Pro?Ile?Phe?Pro?Phe?Met

65 70 75 80

Pro?Pro?Glu?Ala?Val?Asn?Met?Ser?Leu?Gly?Leu?Ser?Leu?Ala?Ala?Ser

85 90 95

Pro?Asn?His?Ser?Gln?Leu?Leu?Ala?Cys?Gly?Pro?Thr?Val?His?Arg?Ala

100 105 110

Cys?Gly?Glu?Asp?Val?Tyr?Ala?Gln?Gly?Phe?Cys?Val?Leu?Leu?Asp?Ala

115 120 125

His?Ala?Gln?Pro?Ile?Gly?Thr?Val?Pro?Ala?Ala?Leu?Pro?Glu?Cys?Pro

130 135 140

Asp?Gln?Glu?Met?Asp?Ile?Val?Phe?Leu?Ile?Asp?Gly?Ser?Gly?Ser?Ile

145 150 155 160

Ser?Ser?Asn?Asp?Phe?Arg?Lys?Met?Lys?Asp?Phe?Val?Arg?Ala?Val?Met

165 170 175

Asp?Gln?Phe?Lys?Asp?Thr?Asn?Thr?Gln?Phe?Ser?Leu?Met?Gln?Tyr?Ser

180 185 190

Asn?Val?Leu?Val?Thr?His?Phe?Thr?Phe?Ser?Ser?Phe?Arg?Asn?Ser?Ser

195 200 205

Asn?Pro?Gln?Gly?Leu?Val?Glu?Pro?Ile?Val?Gln?Leu?Thr?Gly?Leu?Thr

210 215 220

Phe?Thr?Ala?Thr?Gly?Ile?Leu?Lys?Val?Val?Thr?Glu?Leu?Phe?Gln?Thr

225 230 235 240

Lys?Asn?Gly?Ala?Arg?Glu?Ser?Ala?Lys?Lys?Ile?Leu?Ile?Val?Ile?Thr

245 250 255

Asp?Gly?Gln?Lys?Tyr?Lys?Ala?Ala

260

<210>94

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>94

ctggtctgga?ggtgccttcc?tg 22

<210>95

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>95

cctgagcagg?agcacctggc?c 21

<210>96

<211>2499

<212>DNA

＜213〉homo sapiens

<400>96

atgaccttcg?gcactgtgct?tcttctgagt?gtcctggctt?cttatcatgg?attcaacctg?60

gatgtggagg?agcctacgat?cttccaggag?gatgcaggcg?gctttgggca?gagcgtggtg?120

cagttcggtg?gatctcgact?cgtggtggga?gcacccctgg?aggtggtggc?ggccaaccag?180

acgggacggc?tgtatgactg?cgcagctgcc?accggcatgt?gccagcccat?cccgctgcac?240

atccgccctg?aggccgtgaa?catgtccttg?ggcctgaccc?tggcagcctc?caccaacggc?300

tcccggctcc?tggcctgtgg?cccgaccctg?cacagagtct?gtggggagaa?ctcatactca?360

aagggttcct?gcctcctgct?gggctcgcgc?tgggagatca?tccagacagt?ccccgacgcc?420

acgccagagt?gtccacatca?agagatggac?atcgtcttcc?tgattgacgg?ctctggaagc?480

attgaccaaa?atgactttaa?ccagatgaag?ggctttgtcc?aagctgtcat?gggccagttt?540

gagggcactg?acaccctgtt?tgcactgatg?cagtactcaa?acctcctgaa?gatccacttc?600

accttcaccc?aattccggac?cagcccgagc?cagcagagcc?tggtggatcc?catcgtccaa?660

ctgaaaggcc?tgacgttcac?ggccacgggc?atcctgacag?tggtgacaca?gctatttcat?720

cataagaatg?gggcccgaaa?aagtgccaag?aagatcctca?ttgtcatcac?agatgggcag?780

aagtacaaag?accccctgga?atacagtgat?gtcatccccc?aggcagagaa?ggctggcatc?840

atccgctacg?ctatcggggt?gggacacgct?ttccagggac?ccactgccag?gcaggagctg?900

aataccatca?gctcagcgcc?tccgcaggac?cacgtgttca?aggtggacaa?ctttgcagcc?960

cttggcagca?tccagaagca?gctgcaggag?aagatctatg?cagttgaggg?aacccagtcc?1020

agggcaagca?gctccttcca?gcacgagatg?tcccaagaag?gcttcagcac?agccctcaca?1080

atggatggcc?tcttcctggg?ggctgtgggg?agctttagct?ggtctggagg?tgccttcctg?1140

tatcccccaa?atatgagccc?caccttcatc?aacatgtctc?aggagaatgt?ggacatgagg?1200

gactcttacc?tgggttactc?caccgagcta?gccctgtgga?agggggtaca?gaacctggtc?1260

ctgggggccc?cccgctacca?gcataccggg?aaggctgtca?tcttcaccca?ggtgtccagg?1320

caatggagga?agaaggccga?agtcacaggg?acgcagatcg?gctcctactt?cggggcctcc?1380

ctctgctccg?tggatgtgga?cagcgatggc?agcaccgacc?tgatcctcat?tggggccccc?1440

cattactatg?agcagacccg?agggggccag?gtgtccgtgt?gtcccttgcc?tagggggagg?1500

gtgcagtggc?agtgtgacgc?tgttctccgt?ggtgagcagg?gccacccctg?gggccgcttt?1560

ggggcagccc?tgacagtgtt?gggggatgtg?aatgaggaca?agctgataga?cgtggccatt?1620

ggggccccgg?gagagcagga?gaaccggggt?gctgtctacc?tgtttcacgg?agcctcagaa?1680

tccggcatca?gcccctccca?cagccagcgg?attgccagct?cccagctctc?ccccaggctg?1740

cagtattttg?ggcaggcgct?gagtgggggt?caggacctca?cccaggatgg?actgatggac?1800

ctggccgtgg?gggcccgggg?ccaggtgctc?ctgctcagga?gtctgccggt?gctgaaagtg?1860

ggggtggcca?tgagattcag?ccctgtggag?gtggccaagg?ctgtgtaccg?gtgctgggaa?1920

gagaagccca?gtgccctgga?agctggggac?gccaccgtct?gtctcaccat?ccagaaaagc?1980

tcactggacc?agctaggtga?catccaaagc?tctgtcaggt?ttgatctggc?actggaccca?2040

ggtcgtctga?cttctcgtgc?cattttcaat aaa?caaga?accccacttt?gactcgaaga?2100

aaaaccctgg?gactggggat?tcactgtgaa?accctgaagc?tgcttttgcc?agtgaggact?2160

ttgggttctg?ggaaggggga?gagaggagga?gcccaaggct?ggcctggagc?acccccgttc?2220

tctgctgagc?gaggtgggaa?gggttaggat?gttggggctg?gagagaggga?cattagggca?2280

ggagaacctg?gctccacggc?ttggagggag?cactgtcagg?gcagtgggga?gtggatgcag?2340

tggaggagga?cttgtggtgg?agcgtagaga?ggacagcagg?ttcttgaaag?cctgttctct?2400

ctcaggattg?tgtggaggat?gtggtgagcc?ccatcattct?gcacctcaac?ttctcactgg?2460

tgagagagcc?catcccctcc?ccccagaacc?tgcgtcctg 2499

<210>97

<211>3956

<212>DNA

＜213〉homo sapiens

<400>97

tttaactgca?ccaactttaa?aatacgctat?tggagctgga?attaccgcgg?ctgctggcac?60

cagacttgcc?ctccaatgga?tcctcgttaa?aggatttaaa?gtggactcat?tccaattaca?120

gggcctcgaa?agagtcctgt?attgttattt?ttcgtcacta?cctccccggg?tcgggagtgg?180

gtaatttgcg?cgcctgctgc?cttccttgga?tgtggtagcc?gtttctcagg?ctccctctcc?240

ggaatcgaac?cctgattccc?cgtcacccgt?ggtcaccatg?gtaggcacgt?gcagttcggt?300

ggatctcgac?tcgtggtggg?agcacccctg?gaggtggtgg?cggccaacca?gacgggacgg?360

ctgtatgact?gcgcagctgc?caccggcatg?tgccagccca?tcccgctgca?catccgccct?420

gaggccgtga?acatgtcctt?gggcctgacc?ctggcagcct?ccaccaacgg?ctcccggctc?480

ctggcctgtg?gcccgaccct?gcacagagtc?tgtggggaga?actcatactc?aaagggttcc?540

tgcctcctgc?tgggctcgcg?ctgggagatc?atccagacag?tccccgacgc?cacgccagag?600

tgtccacatc?aagagatgga?catcgtcttc?ctgattgacg?gctctggaag?cattgaccaa?660

aatgacttta?accagatgaa?gggctttgtc?caagctgtca?tgggccagtt?tgagggcact?720

gacaccctgt?ttgcactgat?gcagtactca?aacctcctga?agatccactt?caccttcacc?780

caattccgga?ccagcccgag?ccagcagagc?ctggtggatc?ccatcgtcca?actgaaaggc?840

ctgacgttca?cggccacggg?catcctgaca?gtggtgacac?agctatttca?tcataagaat?900

ggggcccgaa?agggtgccaa?gaagatcctc?attgtcatca?cagatgggca?gaagtacaaa?960

gaccccctgg?aatacagtga?tgtcatcccc?caggcagaga?aggctggcat?catccgctac?1020

gctatcgggg?tgggacacgc?tttccaggga?cccactgcca?ggcaggagct?gaataccatc?1080

agctcagcgc?ctccgcagga?ccacgtgttc?aaggtggaca?actttgcagc?ccttggcagc?1140

atccagaagc?agctgcagga?gaagatctat?gcagttgagg?gaacccagtc?cagggcaagc?1200

agctccttcc?agcacgagat?gtcccaagaa?ggcttcagca?cagccctcac?aatggatggc?1260

ctcttcctgg?gggctgtggg?gagctttagc?tggtctggag?gtgccttcct?gtatccccca?1320

aatatgagcc?ccaccttcat?caacatgtct?caggagaatg?tggacatgag?ggactcttac?1380

ctgggttact?ccaccgagct?agccctgtgg?aagggggtac?agaacctggt?cctgggggcc?1440

ccccgctacc?agcataccgg?gaaggctgtc?atcttcaccc?aggtgtccag?gcaatggagg?1500

aagaaggccg?aagtcacagg?gacgcagatc?ggctcctact?tcggggcctc?cctctgctcc?1560

gtggatgtgg?acagcgatgg?cagcaccgac?ctgatcctca?ttggggcccc?ccattactat?1620

gagcagaccc?gagggggcca?ggtgtccgtg?tgtcccttgc?ctagggggag?ggtgcagtgg?1680

cagtgtgacg?ctgttctccg?tggtgagcag?ggccacccct?ggggccgctt?tggggcagcc?1740

ctgacagtgt?tgggggatgt?gaatgaggac?aagctgatag?acgtggccat?tggggccccg?1800

ggagagcagg?agaaccgggg?tgctgtctac?ctgtttcacg?gagcctcaga?atccggcatc?1860

agcccctccc?acagccagcg?gattgccagc?tcccagctct?cccccaggct?gcagtatttt?1920

gggcaggcgc?tgagtggggg?tcaggacctc?acccaggatg?gactgatgga?cctggccgtg?1980

ggggcccggg?gccaggtgct?cctgctcagg?agtctgccgg?tgctgaaagt?gggggtggcc?2040

atgagattca?gccctgtgga?ggtggccaag?gctgtgtacc?ggtgctggga?agagaagccc?2100

agtgccctgg?aagctgggga?cgccaccgtc?tgtctcacca?tccagaaaag?ctcactggac?2160

cagctaggtg?acatccaaag?ctctgtcagg?tttgatctgg?cactggaccc?aggtcgtctg?2220

acttctcgtg?ccattttcaa?tgaaaccaag?aaccccactt?tgactcgaag?aaaaaccctg?2280

ggactgggga?ttcactgtga?aaccctgaag?ctgcttttgc?cagattgtgt?ggaggatgtg?2340

gtgagcccca?tcattctgca?cctcaacttc?tcactggtga?gagagcccat?cccctccccc?2400

cagaacctgc?gtcctgtgct?ggccgtgggc?tcacaagacc?tcttcactgc?ttctctcccc?2460

ttcgagaaga?actgtgggca?agatggcctc?tgtgaagggg?acctgggtgt?caccctcagc?2520

ttctcaggcc?tgcagaccct?gaccgtgggg?agctccctgg?agctcaacgt?gattgtgact?2580

gtgtggaacg?caggtgagga?ttcctacgga?accgtggtca?gcctctacta?tccagcaggg?2640

ctgtcgcacc?gacgggtgtc?aggagcccag?aagcagcccc?atcagagtgc?cctgcgcctg?2700

gcatgtgaga?cagtgcccac?tgaggatgag?ggcctaagaa?gcagccgctg?cagtgtcaac?2760

caccccatct?tccatgaggg?ctctaacggc?accttcatag?tcacattcga?tgtctcctac?2820

aaggccaccc?tgggagacag?gatgcttatg?agggccagtg?caagcagtga?gaacaataag?2880

gcttcaagca?gcaaggccac?cttccagctg?gagctcccgg?tgaagtatgc?agtctacacc?2940

atgatcagca?ggcaggaaga?atccaccaag?tacttcaact?ttgcaacctc?cgatgagaag?3000

aaaatgaaag?aggctgagca?tcgataccgt?gtgaataacc?tcagccagcg?agatctggcc?3060

atcagcatta?acttctgggt?tcctgtcctg?ctgaacgggg?tggctgtgtg?ggatgtggtc?3120

atggaggccc?catctcagag?tctcccctgt?gtttcagaga?gaaaacctcc?ccagcattct?3180

gacttcctga?cccagatttc?aagaagtccc?atgctggact?gctccattgc?tgactgcctg?3240

cagttccgct?gtgacgtccc?ctccttcagc?gtccaggagg?agctggattt?caccctgaag?3300

ggcaatctca?gtttcggctg?ggtccgcgag?acattgcaga?agaaggtgtt?ggtcgtgagt?3360

gtggctgaaa?ttacgttcga?cacatccgtg?tactcccagc?ttccaggaca?ggaggcattt?3420

atgagagctc?agatggagat?ggtgctagaa?gaagacgagg?tctacaatgc?cattcccatc?3480

atcatgggca?gctctgtggg?ggctctgcta?ctgctggcgc?tcatcacagc?cacactgtac?3540

aagcttggct?tcttcaaacg?ccactacaag?gaaatgctgg?aggacaagcc?tgaagacact?3600

gccacattca?gtggggacga?tttcagctgt?gtggccccaa?atgtgccttt?gtcctaataa?3660

tccactttcc?tgtttatctc?taccactgtg?ggctggactt?gcttgcaacc?ataaatcaac?3720

ttacatggaa?acaacttctg?catagatctg?cactggccta?agcaacctac?caggtgctaa?3780

gcaccttctc?ggagagatag?agattgtcaa?tgtttttaca?tatctgtcca?tctttttcag?3840

caatgaccca?ctttttacag?aagcaggcat?ggtgccagca?taaattttca?tatgcttaag?3900

aattgtcaca?tgaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?ctttag 3956

<210>98

<211>3785

<212>DNA

＜213〉homo sapiens

<220>

<221>CDS

<222>(1)..(3483)

<400>98

atg?acc?ttc?ggc?act?gtg?ctt?ctt?ctg?agt?gtc?ctg?gct?tct?tat?cat 48

Met?Thr?Phe?Gly?Thr?Val?Leu?Leu?Leu?Ser?Val?Leu?Ala?Ser?Tyr?His

1 5 10 15

gga?ttc?aac?ctg?gat?gtg?gag?gag?cct?acg?atc?ttc?cag?gag?gat?gca 96

Gly?Phe?Asn?Leu?Asp?Val?Glu?Glu?Pro?Thr?Ile?Phe?Gln?Glu?Asp?Ala

20 25 30

ggc?ggc?ttt?ggg?cag?agc?gtg?gtg?cag?ttc?ggt?gga?tct?cga?ctc?gtg 144

Gly?Gly?Phe?Gly?Gln?Ser?Val?Val?Gln?Phe?Gly?Gly?Ser?Arg?Leu?Val

35 40 45

gtg?gga?gca?ccc?ctg?gag?gtg?gtg?gcg?gcc?aac?cag?acg?gga?cgg?ctg 192

Val?Gly?Ala?Pro?Leu?Glu?Val?Val?Ala?Ala?Asn?Gln?Thr?Gly?Arg?Leu

50 55 60

tat?gac?tgc?gca?gct?gcc?acc?ggc?atg?tgc?cag?ccc?atc?ccg?ctg?cac 240

Tyr?Asp?Cys?Ala?Ala?Ala?Thr?Gly?Met?Cys?Gln?Pro?Ile?Pro?Leu?His

65 70 75 80

atc?cgc?cct?gag?gcc?gtg?aac?atg?tcc?ttg?ggc?ctg?acc?ctg?gca?gcc 288

Ile?Arg?Pro?Glu?Ala?Val?Asn?Met?Ser?Leu?Gly?Leu?Thr?Leu?Ala?Ala

85 90 95

tcc?acc?aac?ggc?tcc?cgg?ctc?ctg?gcc?tgt?ggc?ccg?acc?ctg?cac?aga 336

Ser?Thr?Asn?Gly?Ser?Arg?Leu?Leu?Ala?Cys?Gly?Pro?Thr?Leu?His?Arg

100 105 110

gtc?tgt?ggg?gag?aac?tca?tac?tca?aag?ggt?tcc?tgc?ctc?ctg?ctg?ggc 384

Val?Cys?Gly?Glu?Asn?Ser?Tyr?Ser?Lys?Gly?Ser?Cys?Leu?Leu?Leu?Gly

115 120 125

tcg?cgc?tgg?gag?atc?atc?cag?aca?gtc?ccc?gac?gcc?acg?cca?gag?tgt 432

Ser?Arg?Trp?Glu?Ile?Ile?Gln?Thr?Val?Pro?Asp?Ala?Thr?Pro?Glu?Cys

130 135 140

cca?cat?caa?gag?atg?gac?atc?gtc?ttc?ctg?att?gac?ggc?tct?gga?agc 480

Pro?His?Gln?Glu?Met?Asp?Ile?Val?Phe?Leu?Ile?Asp?Gly?Ser?Gly?Ser

145 150 155 160

att?gac?caa?aat?gac?ttt?aac?cag?atg?aag?ggc?ttt?gtc?caa?gct?gtc 528

Ile?Asp?Gln?Asn?Asp?Phe?Asn?Gln?Met?Lys?Gly?Phe?Val?Gln?Ala?Val

165 170 175

atg?ggc?cag?ttt?gag?ggc?act?gac?acc?ctg?ttt?gca?ctg?atg?cag?tac 576

Met?Gly?Gln?Phe?Glu?Gly?Thr?Asp?Thr?Leu?Phe?Ala?Leu?Met?Gln?Tyr

180 185 190

tca?aac?ctc?ctg?aag?atc?cac?ttc?acc?ttc?acc?caa?ttc?cgg?acc?agc 624

Ser?Asn?Leu?Leu?Lys?Ile?His?Phe?Thr?Phe?Thr?Gln?Phe?Arg?Thr?Ser

195 200 205

ccg?agc?cag?cag?agc?ctg?gtg?gat?ccc?atc?gtc?caa?ctg?aaa?ggc?ctg 672

Pro?Ser?Gln?Gln?Ser?Leu?Val?Asp?Pro?Ile?Val?Gln?Leu?Lys?Gly?Leu

210 215 220

acg?ttc?acg?gcc?acg?ggc?atc?ctg?aca?gtg?gtg?aca?cag?cta?ttt?cat 720

Thr?Phe?Thr?Ala?Thr?Gly?Ile?Leu?Thr?Val?Val?Thr?Gln?Leu?Phe?His

225 230 235 240

cat?aag?aat?ggg?gcc?cga?aaa?agt?gcc?aag?aag?atc?ctc?att?gtc?atc 768

His?Lys?Asn?Gly?Ala?Arg?Lys?Ser?Ala?Lys?Lys?Ile?Leu?Ile?Val?Ile

245 250 255

aca?gat?ggg?cag?aag?tac?aaa?gac?ccc?ctg?gaa?tac?agt?gat?gtc?atc 816

Thr?Asp?Gly?Gln?Lys?Tyr?Lys?Asp?Pro?Leu?Glu?Tyr?Ser?Asp?Val?Ile

260 265 270

ccc?cag?gca?gag?aag?gct?ggc?atc?atc?cgc?tac?gct?atc?ggg?gtg?gga 864

Pro?Gln?Ala?Glu?Lys?Ala?Gly?Ile?Ile?Arg?Tyr?Ala?Ile?Gly?Val?Gly

275 280 285

cac?gct?ttc?cag?gga?ccc?act?gcc?agg?cag?gag?ctg?aat?acc?atc?agc 912

His?Ala?Phe?Gln?Gly?Pro?Thr?Ala?Arg?Gln?Glu?Leu?Asn?Thr?Ile?Ser

290 295 300

tca?gcg?cct?ccg?cag?gac?cac?gtg?ttc?aag?gtg?gac?aac?ttt?gca?gcc 960

Ser?Ala?Pro?Pro?Gln?Asp?His?Val?Phe?Lys?Val?Asp?Asn?Phe?Ala?Ala

305 310 315 320

ctt?ggc?agc?atc?cag?aag?cag?ctg?cag?gag?aag?atc?tat?gca?gtt?gag 1008

Leu?Gly?Ser?Ile?Gln?Lys?Gln?Leu?Gln?Glu?Lys?Ile?Tyr?Ala?Val?Glu

325 330 335

gga?acc?cag?tcc?agg?gca?agc?agc?tcc?ttc?cag?cac?gag?atg?tcc?caa 1056

Gly?Thr?Gln?Ser?Arg?Ala?Ser?Ser?Ser?Phe?Gln?His?Glu?Met?Ser?Gln

340 345 350

gaa?ggc?ttc?agc?aca?gcc?ctc?aca?atg?gat?ggc?ctc?ttc?ctg?ggg?gct 1104

Glu?Gly?Phe?Ser?Thr?Ala?Leu?Thr?Met?Asp?Gly?Leu?Phe?Leu?Gly?Ala

355 360 365

gtg?ggg?agc?ttt?agc?tgg?tct?gga?ggt?gcc?ttc?ctg?tat?ccc?cca?aat 1152

Val?Gly?Ser?Phe?Ser?Trp?Ser?Gly?Gly?Ala?Phe?Leu?Tyr?Pro?Pro?Asn

370 375 380

atg?agc?ccc?acc?ttc?atc?aac?atg?tct?cag?gag?aat?gtg?gac?atg?agg 1200

Met?Ser?Pro?Thr?Phe?Ile?Asn?Met?Ser?Gln?Glu?Asn?Val?Asp?Met?Arg

385 390 395 400

gac?tct?tac?ctg?ggt?tac?tcc?acc?gag?cta?gcc?ctg?tgg?aag?ggg?gta 1248

Asp?Ser?Tyr?Leu?Gly?Tyr?Ser?Thr?Glu?Leu?Ala?Leu?Trp?Lys?Gly?Val

405 410 415

cag?aac?ctg?gtc?ctg?ggg?gcc?ccc?cgc?tac?cag?cat?acc?ggg?aag?gct 1296

Gln?Asn?Leu?Val?Leu?Gly?Ala?Pro?Arg?Tyr?Gln?His?Thr?Gly?Lys?Ala

420 425 430

gtc?atc?ttc?acc?cag?gtg?tcc?agg?caa?tgg?agg?aag?aag?gcc?gaa?gtc 1344

Val?Ile?Phe?Thr?Gln?Val?Ser?Arg?Gln?Trp?Arg?Lys?Lys?Ala?Glu?Val

435 440 445

aca?ggg?acg?cag?atc?ggc?tcc?tac?ttc?ggg?gcc?tcc?ctc?tgc?tcc?gtg 1392

Thr?Gly?Thr?Gln?Ile?Gly?Ser?Tyr?Phe?Gly?Ala?Ser?Leu?Cys?Ser?Val

450 455 460

gat?gtg?gac?agc?gat?ggc?agc?acc?gac?ctg?atc?ctc?att?ggg?gcc?ccc 1440

Asp?Val?Asp?Ser?Asp?Gly?Ser?Thr?Asp?Leu?Ile?Leu?Ile?Gly?Ala?Pro

465 470 475 480

cat?tac?tat?gag?cag?acc?cga?ggg?ggc?cag?gtg?tcc?gtg?tgt?ccc?ttg 1488

His?Tyr?Tyr?Glu?Gln?Thr?Arg?Gly?Gly?Gln?Val?Ser?Val?Cys?Pro?Leu

485 490 495

cct?agg?ggg?agg?gtg?cag?tgg?cag?tgt?gac?gct?gtt?ctc?cgt?ggt?gag 1536

Pro?Arg?Gly?Arg?Val?Gln?Trp?Gln?Cys?Asp?Ala?Val?Leu?Arg?Gly?Glu

500 505 510

cag?ggc?cac?ccc?tgg?ggc?cgc?ttt?ggg?gca?gcc?ctg?aca?gtg?ttg?ggg 1584

Gln?Gly?His?Pro?Trp?Gly?Arg?Phe?Gly?Ala?Ala?Leu?Thr?Val?Leu?Gly

515 520 525

gat?gtg?aat?gag?gac?aag?ctg?ata?gac?gtg?gcc?att?ggg?gcc?ccg?gga 1632

Asp?Val?Asn?Glu?Asp?Lys?Leu?Ile?Asp?Val?Ala?Ile?Gly?Ala?Pro?Gly

530 535 540

gag?cag?gag?aac?cgg?ggt?gct?gtc?tac?ctg?ttt?cac?gga?gcc?tca?gaa 1680

Glu?Gln?Glu?Asn?Arg?Gly?Ala?Val?Tyr?Leu?Phe?His?Gly?Ala?Ser?Glu

545 550 555 560

tcc?ggc?atc?agc?ccc?tcc?cac?agc?cag?cgg?att?gcc?agc?tcc?cag?ctc 1728

Ser?Gly?Ile?Ser?Pro?Ser?His?Ser?Gln?Arg?Ile?Ala?Ser?Ser?Gln?Leu

565 570 575

tcc?ccc?agg?ctg?cag?tat?ttt?ggg?cag?gcg?ctg?agt?ggg?ggt?cag?gac 1776

Ser?Pro?Arg?Leu?Gln?Tyr?Phe?Gly?Gln?Ala?Leu?Ser?Gly?Gly?Gln?Asp

580 585 590

ctc?acc?cag?gat?gga?ctg?atg?gac?ctg?gcc?gtg?ggg?gcc?cgg?ggc?cag 1824

Leu?Thr?Gln?Asp?Gly?Leu?Met?Asp?Leu?Ala?Val?Gly?Ala?Arg?Gly?Gln

595 600 605

gtg?ctc?ctg?ctc?agg?agt?ctg?ccg?gtg?ctg?aaa?gtg?ggg?gtg?gcc?atg 1872

Val?Leu?Leu?Leu?Arg?Ser?Leu?Pro?Val?Leu?Lys?Val?Gly?Val?Ala?Met

610 615 620

aga?ttc?agc?cct?gtg?gag?gtg?gcc?aag?gct?gtg?tac?cgg?tgc?tgg?gaa 1920

Arg?Phe?Ser?Pro?Val?Glu?Val?Ala?Lys?Ala?Val?Tyr?Arg?Cys?Trp?Glu

625 630 635 640

gag?aag?ccc?agt?gcc?ctg?gaa?gct?ggg?gac?gcc?acc?gtc?tgt?ctc?acc 1968

Glu?Lys?Pro?Ser?Ala?Leu?Glu?Ala?Gly?Asp?Ala?Thr?Val?Cys?Leu?Thr

645 650 655

atc?cag?aaa?agc?tca?ctg?gac?cag?cta?ggt?gac?atc?caa?agc?tct?gtc 2016

Ile?Gln?Lys?Ser?Ser?Leu?Asp?Gln?Leu?Gly?Asp?Ile?Gln?Ser?Ser?Val

660 665 670

agg?ttt?gat?ctg?gca?ctg?gac?cca?ggt?cgt?ctg?act?tct?cgt?gcc?att 2064

Arg?Phe?Asp?Leu?Ala?Leu?Asp?Pro?Gly?Arg?Leu?Thr?Ser?Arg?Ala?Ile

675 680 685

ttc?aat?gaa?acc?aag?aac?ccc?act?ttg?act?cga?aga?aaa?acc?ctg?gga 2112

Phe?Asn?Glu?Thr?Lys?Asn?Pro?Thr?Leu?Thr?Arg?Arg?Lys?Thr?Leu?Gly

690 695 700

ctg?ggg?att?cac?tgt?gaa?acc?ctg?aag?ctg?ctt?ttg?cca?gat?tgt?gtg 2160

Leu?Gly?Ile?His?Cys?Glu?Thr?Leu?Lys?Leu?Leu?Leu?Pro?Asp?Cys?Val

705 710 715 720

gag?gat?gtg?gtg?agc?ccc?atc?att?ctg?cac?ctc?aac?ttc?tca?ctg?gtg 2208

Glu?Asp?Val?Val?Ser?Pro?Ile?Ile?Leu?His?Leu?Asn?Phe?Ser?Leu?Val

725 730 735

aga?gag?ccc?atc?ccc?tcc?ccc?cag?aac?ctg?cgt?cct?gtg?ctg?gcc?gtg 2256

Arg?Glu?Pro?Ile?Pro?Ser?Pro?Gln?Asn?Leu?Arg?Pro?Val?Leu?Ala?Val

740 745 750

ggc?tca?caa?gac?ctc?ttc?act?gct?tct?ctc?ccc?ttc?gag?aag?aac?tgt 2304

Gly?Ser?Gln?Asp?Leu?Phe?Thr?Ala?Ser?Leu?Pro?Phe?Glu?Lys?Asn?cys

755 760 765

ggg?caa?gat?ggc?ctc?tgt?gaa?ggg?gac?ctg?ggt?gtc?acc?ctc?agc?ttc 2352

Gly?Gln?Asp?Gly?Leu?Cys?Glu?Gly?Asp?Leu?Gly?Val?Thr?Leu?Ser?Phe

770 775 780

tca?ggc?ctg?cag?acc?ctg?acc?gtg?ggg?agc?tcc?ctg?gag?ctc?aac?gtg 2400

Ser?Gly?Leu?Gln?Thr?Leu?Thr?Val?Gly?Ser?Ser?Leu?Glu?Leu?Asn?Val

785 790 795 800

att?gtg?act?gtg?tgg?aac?gca?ggt?gag?gat?tcc?tac?gga?acc?gtg?gtc 2448

Ile?Val?Thr?Val?Trp?Asn?Ala?Gly?Glu?Asp?Ser?Tyr?Gly?Thr?Val?Val

805 810 815

agc?ctc?tac?tat?cca?gca?ggg?ctg?tcg?cac?cga?cgg?gtg?tca?gga?gcc 2496

Ser?Leu?Tyr?Tyr?Pro?Ala?Gly?Leu?Ser?His?Arg?Arg?Val?Ser?Gly?Ala

820 825 830

cag?aag?cag?ccc?cat?cag?agt?gcc?ctg?cgc?ctg?gca?tgt?gag?aca?gtg 2544

Gln?Lys?Gln?Pro?His?Gln?Ser?Ala?Leu?Arg?Leu?Ala?Cys?Glu?Thr?Val

835 840 845

ccc?act?gag?gat?gag?ggc?cta?aga?agc?agc?cgc?tgc?agt?gtc?aac?cac 2592

Pro?Thr?Glu?Asp?Glu?Gly?Leu?Arg?Ser?Ser?Arg?Cys?Ser?Val?Asn?His

850 855 860

ccc?atc?ttc?cat?gag?ggc?tct?aac?ggc?acc?ttc?ata?gtc?aca?ttc?gat 2640

Pro?Ile?Phe?His?Glu?Gly?Ser?Asn?Gly?Thr?Phe?Ile?Val?Thr?Phe?Asp

865 870 875 880

gtc?tcc?tac?aag?gcc?acc?ctg?gga?gac?agg?atg?ctt?atg?agg?gcc?agt 2688

Val?Ser?Tyr?Lys?Ala?Thr?Leu?Gly?Asp?Arg?Met?Leu?Met?Arg?Ala?Ser

885 890 895

gca?agc?agt?gag?aac?aat?aag?gct?tca?agc?agc?aag?gcc?acc?ttc?cag 2736

Ala?Ser?Ser?Glu?Asn?Asn?Lys?Ala?Ser?Ser?Ser?Lys?Ala?Thr?Phe?Gln

900 905 910

ctg?gag?ctc?ccg?gtg?aag?tat?gca?gtc?tac?acc?atg?atc?agc?agg?cag 2784

Leu?Glu?Leu?Pro?Val?Lys?Tyr?Ala?Val?Tyr?Thr?Met?Ile?Ser?Arg?Gln

915 920 925

gaa?gaa?tcc?acc?aag?tac?ttc?aac?ttt?gca?acc?tcc?gat?gag?aag?aaa 2832

Glu?Glu?Ser?Thr?Lys?Tyr?Phe?Asn?Phe?Ala?Thr?Ser?Asp?Glu?Lys?Lys

930 935 940

atg?aaa?gag?gct?gag?cat?cga?tac?cgt?gtg?aat?aac?ctc?agc?cag?cga 2880

Met?Lys?Glu?Ala?Glu?His?Arg?Tyr?Arg?Val?Asn?Asn?Leu?Ser?Gln?Arg

945 950 955 960

gat?ctg?gcc?atc?agc?att?aac?ttc?tgg?gtt?cct?gtc?ctg?ctg?aac?ggg 2928

Asp?Leu?Ala?Ile?Ser?Ile?Asn?Phe?Trp?Val?Pro?Val?Leu?Leu?Asn?Gly

965 970 975

gtg?gct?gtg?tgg?gat?gtg?gtc?atg?gag?gcc?cca?tct?cag?agt?ctc?ccc 2976

Val?Ala?Val?Trp?Asp?Val?Val?Met?Glu?Ala?Pro?Ser?Gln?Ser?Leu?Pro

980 985 990

tgt?gtt?tca?gag?aga?aaa?cct?ccc?cag?cat?tct?gac?ttc?ctg?acc?cag 3024

Cys?Val?Ser?Glu?Arg?Lys?Pro?Pro?Gln?His?Ser?Asp?Phe?Leu?Thr?Gln

995 1000 1005

att?tca?aga?agt?ccc?atg?ctg?gac?tgc?tcc?att?gct?gac?tgc?ctg?cag 3072

Ile?Ser?Arg?Ser?Pro?Met?Leu?Asp?Cys?Ser?Ile?Ala?Asp?Cys?Leu?Gln

1010 1015 1020

ttc?cgc?tgt?gac?gtc?ccc?tcc?ttc?agc?gtc?cag?gag?gag?ctg?gat?ttc 3120

Phe?Arg?Cys?Asp?Val?Pro?Ser?Phe?Ser?Val?Gln?Glu?Glu?Leu?Asp?Phe

1025 1030 1035 1040

acc?ctg?aag?ggc?aat?ctc?agt?ttc?ggc?tgg?gtc?cgc?gag?aca?ttg?cag 3168

Thr?Leu?Lys?Gly?Asn?Leu?Ser?Phe?Gly?Trp?Val?Arg?Glu?Thr?Leu?Gln

1045 1050 1055

aag?aag?gtg?ttg?gtc?gtg?agt?gtg?gct?gaa?att?acg?ttc?gac?aca?tcc 3216

Lys?Lys?Val?Leu?Val?Val?Ser?Val?Ala?Glu?Ile?Thr?Phe?Asp?Thr?Ser

1060 1065 1070

gtg?tac?tcc?cag?ctt?cca?gga?cag?gag?gca?ttt?atg?aga?gct?cag?atg 3264

Val?Tyr?Ser?Gln?Leu?Pro?Gly?Gln?Glu?Ala?Phe?Met?Arg?Ala?Gln?Met

1075 1080 1085

gag?atg?gtg?cta?gaa?gaa?gac?gag?gtc?tac?aat?gcc?att?ccc?atc?atc 3312

Glu?Met?Val?Leu?Glu?Glu?Asp?Glu?Val?Tyr?Asn?Ala?Ile?Pro?Ile?Ile

1090 1095 1100

atg?ggc?agc?tct?gtg?ggg?gct?ctg?cta?ctg?ctg?gcg?ctc?atc?aca?gcc 3360

Met?Gly?Ser?Ser?Val?Gly?Ala?Leu?Leu?Leu?Leu?Ala?Leu?Ile?Thr?Ala

1105 1110 1115 1120

aca?ctg?tac?aag?ctt?ggc?ttc?ttc?aaa?cgc?cac?tac?aag?gaa?atg?ctg 3408

Thr?Leu?Tyr?Lys?Leu?Gly?Phe?Phe?Lys?Arg?His?Tyr?Lys?Glu?Met?Leu

1125 1130 1135

gag?gac?aag?cct?gaa?gac?act?gcc?aca?ttc?agt?ggg?gac?gat?ttc?agc 3456

Glu?Asp?Lys?Pro?Glu?Asp?Thr?Ala?Thr?Phe?Ser?Gly?Asp?Asp?Phe?Ser

1140 1145 1150

tgt?gtg?gcc?cca?aat?gtg?cct?ttg?tcc?taataatcca?ctttcctgtt 3503

Cys?Val?Ala?Pro?Asn?Val?Pro?Leu?Ser

1155 1160

tatctctacc?actgtgggct?ggacttgctt?gcaaccataa?atcaacttac?atggaaacaa?3563

cttctgcata?gatctgcact?ggcctaagca?acctaccagg?tgctaagcac?cttctcggag?3623

agatagagat?tgtcaatgtt?tttacatatc?tgtccatctt?tttcagcaat?gacccacttt?3683

ttacagaagc?aggcatggtg?ccagcataaa?ttttcatatg?cttaagaatt?gtcacatgaa?3743

aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaacttt?ag 3785

<210>99

<211>1161

<212>PRT

＜213〉homo sapiens

<400>99

Met?Thr?Phe?Gly?Thr?Val?Leu?Leu?Leu?Ser?Val?Leu?Ala?Ser?Tyr?His

1 5 10 15

Gly?Phe?Asn?Leu?Asp?Val?Glu?Glu?Pro?Thr?Ile?Phe?Gln?Glu?Asp?Ala

20 25 30

Gly?Gly?Phe?Gly?Gln?Ser?Val?Val?Gln?Phe?Gly?Gly?Ser?Arg?Leu?Val

35 40 45

Val?Gly?Ala?Pro?Leu?Glu?Val?Val?Ala?Ala?Asn?Gln?Thr?Gly?Arg?Leu

50 55 60

Tyr?Asp?Cys?Ala?Ala?Ala?Thr?Gly?Met?Cys?Gln?Pro?Ile?Pro?Leu?His

65 70 75 80

Ile?Arg?Pro?Glu?Ala?Val?Asn?Met?Ser?Leu?Gly?Leu?Thr?Leu?Ala?Ala

85 90 95

Ser?Thr?Asn?Gly?Ser?Arg?Leu?Leu?Ala?Cys?Gly?Pro?Thr?Leu?His?Arg

100 105 110

Val?Cys?Gly?Glu?Asn?Ser?Tyr?Ser?Lys?Gly?Ser?Cys?Leu?Leu?Leu?Gly

115 120 125

Ser?Arg?Trp?Glu?Ile?Ile?Gln?Thr?Val?Pro?Asp?Ala?Thr?Pro?Glu?Cys

130 135 140

Pro?His?Gln?Glu?Met?Asp?Ile?Val?Phe?Leu?Ile?Asp?Gly?Ser?Gly?Ser

145 150 155 160

Ile?Asp?Gln?Asn?Asp?Phe?Asn?Gln?Met?Lys?Gly?Phe?Val?Gln?Ala?Val

165 170 175

Met?Gly?Gln?Phe?Glu?Gly?Thr?Asp?Thr?Leu?Phe?Ala?Leu?Met?Gln?Tyr

180 185 190

Ser?Asn?Leu?Leu?Lys?Ile?His?Phe?Thr?Phe?Thr?Gln?Phe?Arg?Thr?Ser

195 200 205

Pro?Ser?Gln?Gln?Ser?Leu?Val?Asp?Pro?Ile?Val?Gln?Leu?Lys?Gly?Leu

210 215 220

Thr?Phe?Thr?Ala?Thr?Gly?Ile?Leu?Thr?Val?Val?Thr?Gln?Leu?Phe?His

225 230 235 240

His?Lys?Asn?Gly?Ala?Arg?Lys?Ser?Ala?Lys?Lys?Ile?Leu?Ile?Val?Ile

245 250 255

Thr?Asp?Gly?Gln?Lys?Tyr?Lys?Asp?Pro?Leu?Glu?Tyr?Ser?Asp?Val?Ile

260 265 270

Pro?Gln?Ala?Glu?Lys?Ala?Gly?Ile?Ile?Arg?Tyr?Ala?Ile?Gly?Val?Gly

275 280 285

His?Ala?Phe?Gln?Gly?Pro?Thr?Ala?Arg?Gln?Glu?Leu?Asn?Thr?Ile?Ser

290 295 300

Ser?Ala?Pro?Pro?Gln?Asp?His?Val?Phe?Lys?Val?Asp?Asn?Phe?Ala?Ala

305 310 315 320

Leu?Gly?Ser?Ile?Gln?Lys?Gln?Leu?Gln?Glu?Lys?Ile?Tyr?Ala?Val?Glu

325 330 335

Gly?Thr?Gln?Ser?Arg?Ala?Ser?Ser?Ser?Phe?Gln?His?Glu?Met?Ser?Gln

340 345 350

Glu?Gly?Phe?Ser?Thr?Ala?Leu?Thr?Met?Asp?Gly?Leu?Phe?Leu?Gly?Ala

355 360 365

Val?Gly?Ser?Phe?Ser?Trp?Ser?Gly?Gly?Ala?Phe?Leu?Tyr?Pro?Pro?Asn

370 375 380

Met?Ser?Pro?Thr?Phe?Ile?Asn?Met?Ser?Gln?Glu?Asn?Val?Asp?Met?Arg

385 390 395 400

Asp?Ser?Tyr?Leu?Gly?Tyr?Ser?Thr?Glu?Leu?Ala?Leu?Trp?Lys?Gly?Val

405 410 415

Gln?Asn?Leu?Val?Leu?Gly?Ala?Pro?Arg?Tyr?Gln?His?Thr?Gly?Lys?Ala

420 425 430

Val?Ile?Phe?Thr?Gln?Val?Ser?Arg?Gln?Trp?Arg?Lys?Lys?Ala?Glu?Val

435 440 445

Thr?Gly?Thr?Gln?Ile?Gly?Ser?Tyr?Phe?Gly?Ala?Ser?Leu?Cys?Ser?Val

450 455 460

Asp?Val?Asp?Ser?Asp?Gly?Ser?Thr?Asp?Leu?Ile?Leu?Ile?Gly?Ala?Pro

465 470 475 480

His?Tyr?Tyr?Glu?Gln?Thr?Arg?Gly?Gly?Gln?Val?Ser?Val?Cys?Pro?Leu

485 490 495

Pro?Arg?Gly?Arg?Val?Gln?Trp?Gln?Cys?Asp?Ala?Val?Leu?Arg?Gly?Glu

500 505 510

Gln?Gly?His?Pro?Trp?Gly?Arg?Phe?Gly?Ala?Ala?Leu?Thr?Val?Leu?Gly

515 520 525

Asp?Val?Asn?Glu?Asp?Lys?Leu?Ile?Asp?Val?Ala?Ile?Gly?Ala?Pro?Gly

530 535 540

Glu?Gln?Glu?Asn?Arg?Gly?Ala?Val?Tyr?Leu?Phe?His?Gly?Ala?Ser?Glu

545 550 555 560

Ser?Gly?Ile?Ser?Pro?Ser?His?Ser?Gln?Arg?Ile?Ala?Ser?Ser?Gln?Leu

565 570 575

Ser?Pro?Arg?Leu?Gln?Tyr?Phe?Gly?Gln?Ala?Leu?Ser?Gly?Gly?Gln?Asp

580 585 590

Leu?Thr?Gln?Asp?Gly?Leu?Met?Asp?Leu?Ala?Val?Gly?Ala?Arg?Gly?Gln

595 600 605

Val?Leu?Leu?Leu?Arg?Ser?Leu?Pro?Val?Leu?Lys?Val?Gly?Val?Ala?Met

610 615 620

Arg?Phe?Ser?Pro?Val?Glu?Val?Ala?Lys?Ala?Val?Tyr?Arg?Cys?Trp?Glu

625 630 635 640

Glu?Lys?Pro?Ser?Ala?Leu?Glu?Ala?Gly?Asp?Ala?Thr?Val?Cys?Leu?Thr

645 650 655

Ile?Gln?Lys?Ser?Ser?Leu?Asp?Gln?Leu?Gly?Asp?Ile?Gln?Ser?Ser?Val

660 665 670

Arg?Phe?Asp?Leu?Ala?Leu?Asp?Pro?Gly?Arg?Leu?Thr?Ser?Arg?Ala?Ile

675 680 685

Phe?Asn?Glu?Thr?Lys?Asn?Pro?Thr?Leu?Thr?Arg?Arg?Lys?Thr?Leu?Gly

690 695 700

Leu?Gly?Ile?His?Cys?Glu?Thr?Leu?Lys?Leu?Leu?Leu?Pro?Asp?Cys?Val

705 710 715 720

Glu?Asp?Val?Val?Ser?Pro?Ile?Ile?Leu?His?Leu?Asn?Phe?Ser?Leu?Val

725 730 735

Arg?Glu?Pro?Ile?Pro?Ser?Pro?Gln?Asn?Leu?Arg?Pro?Val?Leu?Ala?Val

740 745 750

Gly?Ser?Gln?Asp?Leu?Phe?Thr?Ala?Ser?Leu?Pro?Phe?Glu?Lys?Asn?Cys

755 760 765

Gly?Gln?Asp?Gly?Leu?Cys?Glu?Gly?Asp?Leu?Gly?Val?Thr?Leu?Ser?Phe

770 775 780

Ser?Gly?Leu?Gln?Thr?Leu?Thr?Val?Gly?Ser?Ser?Leu?Glu?Leu?Asn?Val

785 790 795 800

Ile?Val?Thr?Val?Trp?Asn?Ala?Gly?Glu?Asp?Ser?Tyr?Gly?Thr?Val?Val

805 810 815

Ser?Leu?Tyr?Tyr?Pro?Ala?Gly?Leu?Ser?His?Arg?Arg?Val?Ser?Gly?Ala

820 825 830

Gln?Lys?Gln?Pro?His?Gln?Ser?Ala?Leu?Arg?Leu?Ala?Cys?Glu?Thr?Val

835 840 845

Pro?Thr?Glu?Asp?Glu?Gly?Leu?Arg?Ser?Ser?Arg?Cys?Ser?Val?Asn?His

850 855 860

Pro?Ile?Phe?His?Glu?Gly?Ser?Asn?Gly?Thr?Phe?Ile?Val?Thr?Phe?Asp

865 870 875 880

Val?Ser?Tyr?Lys?Ala?Thr?Leu?Gly?Asp?Arg?Met?Leu?Met?Arg?Ala?Ser

885 890 895

Ala?Ser?Ser?Glu?Asn?Asn?Lys?Ala?Ser?Ser?Ser?Lys?Ala?Thr?Phe?Gln

900 905 910

Leu?Glu?Leu?Pro?Val?Lys?Tyr?Ala?Val?Tyr?Thr?Met?Ile?Ser?Arg?Gln

915 920 925

Glu?Glu?Ser?Thr?Lys?Tyr?Phe?Asn?Phe?Ala?Thr?Ser?Asp?Glu?Lys?Lys

930 935 940

Met?Lys?Glu?Ala?Glu?His?Arg?Tyr?Arg?Val?Asn?Asn?Leu?Ser?Gln?Arg

945 950 955 960

Asp?Leu?Ala?Ile?Ser?Ile?Asn?Phe?Trp?Val?Pro?Val?Leu?Leu?Asn?Gly

965 970 975

Val?Ala?Val?Trp?Asp?Val?Val?Met?Glu?Ala?Pro?Ser?Gln?Ser?Leu?Pro

980 985 990

Cys?Val?Ser?Glu?Arg?Lys?Pro?Pro?Gln?His?Ser?Asp?Phe?Leu?Thr?Gln

995 1000 1005

Ile?Ser?Arg?Ser?Pro?Met?Leu?Asp?Cys?Ser?Ile?Ala?Asp?Cys?Leu?Gln

1010 1015 1020

Phe?Arg?Cys?Asp?Val?Pro?Ser?Phe?Ser?Val?Gln?Glu?Glu?Leu?Asp?Phe

1025 1030 1035 1040

Thr?Leu?Lys?Gly?Asn?Leu?Ser?Phe?Gly?Trp?Val?Arg?Glu?Thr?Leu?Gln

1045 1050 1055

Lys?Lys?Val?Leu?Val?Val?Ser?Val?Ala?Glu?Ile?Thr?Phe?Asp?Thr?Ser

1060 1065 1070

Val?Tyr?Ser?Gln?Leu?Pro?Gly?Gln?Glu?Ala?Phe?Met?Arg?Ala?Gln?Met

1075 1080 1085

Glu?Met?Val?Leu?Glu?Glu?Asp?Glu?Val?Tyr?Asn?Ala?Ile?Pro?Ile?Ile

1090 1095 1100

Met?Gly?Ser?Ser?Val?Gly?Ala?Leu?Leu?Leu?Leu?Ala?Leu?Ile?Thr?Ala

1105 1110 1115 1120

Thr?Leu?Tyr?Lys?Leu?Gly?Phe?Phe?Lys?Arg?His?Tyr?Lys?Glu?Met?Leu

1125 1130 1135

Glu?Asp?Lys?Pro?Glu?Asp?Thr?Ala?Thr?Phe?Ser?Gly?Asp?Asp?Phe?Ser

1140 1145 1150

Cys?Val?Ala?Pro?Asn?Val?Pro?Leu?Ser

1155 1160

<210>100

<211>1318

<212>DNA

＜213〉rabbit

<220>

<22l>CDS

<222>(17)..(1255)

<400>100

aattcggcac?gagctt?ggg?gct?gtg?gtc?ctc?ctt?ggg?gtc?ctg?gct?tct?tac?52

Gly?Ala?Val?Val?Leu?Leu?Gly?Val?Leu?Ala?Ser?Tyr

1 5 10

cac?gga?ttc?aac?ttg?gac?gtg?gat?gag?ccg?gtg?atc?ttc?cag?gaa?gac 100

His?Gly?Phe?Asn?Leu?Asp?Val?Asp?Glu?Pro?Val?Ile?Phe?Gln?Glu?Asp

15 20 25

gca?gcg?ggc?ttc?ggg?cag?agc?gtg?atg?cag?ttt?gga?gga?tct?cga?ctc 148

Ala?Ala?Gly?Phe?Gly?Gln?Ser?Val?Met?Gln?Phe?Gly?Gly?Ser?Arg?Leu

30 35 40

gtg?gtg?gga?gcc?ccc?ctg?gcg?gtg?gtg?tcg?gcc?aac?cac?aca?gga?cgg 196

Val?Val?Gly?Ala?Pro?Leu?Ala?Val?Val?Ser?Ala?Asn?His?Thr?Gly?Arg

45 50 55 60

ctg?tac?gag?tgt?gcg?cct?gcc?tcc?ggc?acc?tgc?acg?ccc?att?ttc?cca 244

Leu?Tyr?Glu?Cys?Ala?Pro?Ala?Ser?Gly?Thr?Cys?Thr?Pro?Ile?Phe?Pro

65 70 75

ttc?atg?ccc?ccc?gaa?gcc?gtg?aac?atg?tcc?ctg?ggc?ctg?tcc?ctg?gca 292

Phe?Met?Pro?Pro?Glu?Ala?Val?Asn?Met?Ser?Leu?Gly?Leu?Ser?Leu?Ala

80 85 90

gcc?tcc?ccc?aac?cat?tcc?cag?ctg?ctg?gct?tgt?ggc?ccg?acc?gtg?cat 340

Ala?Ser?Pro?Asn?His?Ser?Gln?Leu?Leu?Ala?Cys?Gly?Pro?Thr?Val?His

95 100 105

aga?gcc?tgc?ggg?gag?gac?gtg?tac?gcc?cag?ggt?ttc?tgt?gtg?ctg?ctg 388

Arg?Ala?Cys?Gly?Glu?Asp?Val?Tyr?Ala?Gln?Gly?Phe?Cys?Val?Leu?Leu

110 115 120

gat?gcc?cac?gca?cag?ccc?atc?ggg?act?gtg?cca?gct?gcc?ctg?ccc?gag 436

Asp?Ala?His?Ala?Gln?Pro?Ile?Gly?Thr?Val?Pro?Ala?Ala?Leu?Pro?Glu

125 130 135 140

tgc?cca?gat?caa?gag?atg?gac?att?gtc?ttc?ctg?att?gac?ggc?tct?ggc 484

Cys?Pro?Asp?Gln?Glu?Met?Asp?Ile?Val?Phe?Leu?Ile?Asp?Gly?Ser?Gly

145 150 155

agc?att?agc?tca?aat?gac?ttc?cgc?aag?atg?aag?gac?ttt?gtc?aga?gct 532

Ser?Ile?Ser?Ser?Asn?Asp?Phe?Arg?Lys?Met?Lys?Asp?Phe?Val?Arg?Ala

160 165 170

gtg?atg?gac?cag?ttc?aag?gac?acc?aac?acc?cag?ttc?tcg?ctg?atg?cag 580

Val?Met?Asp?Gln?Phe?Lys?Asp?Thr?Asn?Thr?Gln?Phe?Ser?Leu?Met?Gln

175 180 185

tac?tcc?aat?gtg?ctg?gtg?aca?cat?ttc?acc?ttc?agc?agc?ttc?cgg?aac 628

Tyr?Ser?Asn?Val?Leu?Val?Thr?His?Phe?Thr?Phe?Ser?Ser?Phe?Arg?Asn

190 195 200

agc?tcc?aat?cct?cag?ggc?cta?gtg?gag?ccc?att?gtg?cag?ctg?aca?ggc 676

Ser?Ser?Asn?Pro?Gln?Gly?Leu?Val?Glu?Pro?Ile?Val?Gln?Leu?Thr?Gly

205 210 215 220

ctc?acg?ttc?acg?gcc?aca?ggg?atc?ctg?aaa?gtg?gtg?aca?gag?ctg?ttt 724

Leu?Thr?Phe?Thr?Ala?Thr?Gly?Ile?Leu?Lys?Val?Val?Thr?Glu?Leu?Phe

225 230 235

caa?acc?aag?aac?ggg?gcc?cgc?gaa?agt?gcc?aag?aag?atc?ctc?atc?gtc 772

Gln?Thr?Lys?Asn?Gly?Ala?Arg?Glu?Ser?Ala?Lys?Lys?Ile?Leu?Ile?Val

240 245 250

atc?aca?gat?ggg?cag?aag?tac?aaa?gac?ccc?ctg?cac?tac?agt?gct?gtc 820

Ile?Thr?Asp?Gly?Gln?Lys?Tyr?Lys?Asp?Pro?Leu?His?Tyr?Ser?Ala?Val

255 260 265

atc?cca?cag?gca?gag?cag?gcg?ggc?atc?atc?cgc?tac?gcc?atc?ggg?gtg 868

Ile?Pro?Gln?Ala?Glu?Gln?Ala?Gly?Ile?Ile?Arg?Tyr?Ala?Ile?Gly?Val

270 275 280

ggg?gac?gcg?ttc?cag?aaa?ccc?aca?gcc?agg?cag?gag?ctg?gac?acc?atc 916

Gly?Asp?Ala?Phe?Gln?Lys?Pro?Thr?Ala?Arg?Gln?Glu?Leu?Asp?Thr?Ile

285 290 295 300

gcc?tcc?gag?ccg?ccc?gac?gcc?cac?gtg?ttc?cag?gtg?gac?aat?ttc?tca 964

Ala?Ser?Glu?Pro?Pro?Asp?Ala?His?Val?Phe?Gln?Val?Asp?Asn?Phe?Ser

305 310 315

gca?ctc?agc?agc?atc?caa?aag?cag?ctg?tat?gac?agg?atc?ttt?gcc?gtc 1012

Ala?Leu?Ser?Ser?Ile?Gln?Lys?Gln?Leu?Tyr?Asp?Arg?Ile?Phe?Ala?Val

320 325 330

gag?gga?acc?ctg?tca?tcg?gca?agc?acc?tcc?ttc?cag?cat?gag?atg?tcc 1060

Glu?Gly?Thr?Leu?Ser?Ser?Ala?Ser?Thr?Ser?Phe?Gln?His?Glu?Met?Ser

335 340 345

caa?gag?ggc?ttc?agc?tca?ctt?ctc?acc?acg?gaa?gga?ccg?gtg?ctg?ggg 1108

Gln?Glu?Gly?Phe?Ser?Ser?Leu?Leu?Thr?Thr?Glu?Gly?Pro?Val?Leu?Gly

350 355 360

gct?gtg?ggc?agc?ttc?gat?tgg?tcc?ggg?ggt?gct?ttc?ctg?tac?ccc?ccc 1156

Ala?Val?Gly?Ser?Phe?Asp?Trp?Ser?Gly?Gly?Ala?Phe?Leu?Tyr?Pro?Pro

365 370 375 380

ggc?ggg?agc?ccc?acc?ttc?atc?aac?atg?tct?cag?cag?aac?gtg?gac?atg 1204

Gly?Gly?Ser?Pro?Thr?Phe?Ile?Asn?Met?Ser?Gln?Gln?Asn?Val?Asp?Met

385 390 395

agg?gac?tcc?tac?ctg?ggt?gag?gaa?ggg?gtg?ggg?gtg?ggg?aca?ggt?ggg 1252

Arg?Asp?Ser?Tyr?Leu?Gly?Glu?Glu?Gly?Val?Gly?Val?Gly?Thr?Gly?Gly

400 405 410

agc?tgaggcttgg?ggtggggtgg?ggctgggctg?ggaggggagg?gaagaggagg 1305

Ser

ggagaggcaa?aga 1318

<210>101

<211>413

<212>PRT

＜213〉rabbit

<400>101

Gly?Ala?Val?Val?Leu?Leu?Gly?Val?Leu?Ala?Ser?Tyr?His?Gly?Phe?Asn

1 5 10 15

Leu?Asp?Val?Asp?Glu?Pro?Val?Ile?Phe?Gln?Glu?Asp?Ala?Ala?Gly?Phe

20 25 30

Gly?Gln?Ser?Val?Met?Gln?Phe?Gly?Gly?Ser?Arg?Leu?Val?Val?Gly?Ala

35 40 45

Pro?Leu?Ala?Val?Val?Ser?Ala?Asn?His?Thr?Gly?Arg?Leu?Tyr?Glu?Cys

50 55 60

Ala?Pro?Ala?Ser?Gly?Thr?Cys?Thr?Pro?Ile?Phe?Pro?Phe?Met?Pro?Pro

65 70 75 80

Glu?Ala?Val?Asn?Met?Ser?Leu?Gly?Leu?Ser?Leu?Ala?Ala?Ser?Pro?Asn

85 90 95

His?Ser?Gln?Leu?Leu?Ala?Cys?Gly?Pro?Thr?Val?His?Arg?Ala?Cys?Gly

100 105 110

Glu?Asp?Val?Tyr?Ala?Gln?Gly?Phe?Cys?Val?Leu?Leu?Asp?Ala?His?Ala

115 120 125

Gln?Pro?Ile?Gly?Thr?Val?Pro?Ala?Ala?Leu?Pro?Glu?Cys?Pro?Asp?Gln

130 135 140

Glu?Met?Asp?Ile?Val?Phe?Leu?Ile?Asp?Gly?Ser?Gly?Ser?Ile?Ser?Ser

145 150 155 160

Asn?Asp?Phe?Arg?Lys?Met?Lys?Asp?Phe?Val?Arg?Ala?Val?Met?Asp?Gln

165 170 175

Phe?Lys?Asp?Thr?Asn?Thr?Gln?Phe?Ser?Leu?Met?Gln?Tyr?Ser?Asn?Val

180 185 190

Leu?Val?Thr?His?Phe?Thr?Phe?Ser?Ser?Phe?Arg?Asn?Ser?Ser?Asn?Pro

195 200 205

Gln?Gly?Leu?Val?Glu?Pro?Ile?Val?Gln?Leu?Thr?Gly?Leu?Thr?Phe?Thr

210 215 220

Ala?Thr?Gly?Ile?Leu?Lys?Val?Val?Thr?Glu?Leu?Phe?Gln?Thr?Lys?Asn

225 230 235 240

Gly?Ala?Arg?Glu?Ser?Ala?Lys?Lys?Ile?Leu?Ile?Val?Ile?Thr?Asp?Gly

245 250 255

Gln?Lys?Tyr?Lys?Asp?Pro?Leu?His?Tyr?Ser?Ala?Val?Ile?Pro?Gln?Ala

260 265 270

Glu?Gln?Ala?Gly?Ile?Ile?Arg?Tyr?Ala?Ile?Gly?Val?Gly?Asp?Ala?Phe

275 280 285

Gln?Lys?Pro?Thr?Ala?Arg?Gln?Glu?Leu?Asp?Thr?Ile?Ala?Ser?Glu?Pro

290 295 300

Pro?Asp?Ala?His?Val?Phe?Gln?Val?Asp?Asn?Phe?Ser?Ala?Leu?Ser?Ser

305 310 315 320

Ile?Gln?Lys?Gln?Leu?Tyr?Asp?Arg?Ile?Phe?Ala?Val?Glu?Gly?Thr?Leu

325 330 335

Ser?Ser?Ala?Ser?Thr?Ser?Phe?Gln?His?Glu?Met?Ser?Gln?Glu?Gly?Phe

340 345 350

Ser?Ser?Leu?Leu?Thr?Thr?Glu?Gly?Pro?Val?Leu?Gly?Ala?Val?Gly?Ser

355 360 365

Phe?Asp?Trp?Ser?Gly?Gly?Ala?Phe?Leu?Tyr?Pro?Pro?Gly?Gly?Ser?Pro

370 375 380

Thr?Phe?Ile?Asn?Met?Ser?Gln?Gln?Asn?Val?Asp?Met?Arg?Asp?Ser?Tyr

385 390 395 400

Leu?Gly?Glu?Glu?Gly?Val?Gly?Val?Gly?Thr?Gly?Gly?Ser

405 410

<210>102

<211>1484

<212>DNA

＜213〉rabbit

<220>

<221>CDS

<222>(1)..(1482)

<400>102

gat?gtc?cag?agc?tcc?atc?agc?tat?gat?ctg?gca?ctg?gac?cca?ggc?cgc 48

Asp?Val?Gln?Ser?Ser?Ile?Ser?Tyr?Asp?Leu?Ala?Leu?Asp?Pro?Gly?Arg

1 5 10 15

ctg?gtc?tct?cgg?gcc?att?ttt?caa?gag?acc?cag?aac?cag?act?tta?act 96

Leu?Val?Ser?Arg?Ala?Ile?Phe?Gln?Glu?Thr?Gln?Asn?Gln?Thr?Leu?Thr

20 25 30

cga?agg?aag?acc?ctg?ggg?ctg?ggg?cgt?cac?tgt?gaa?acc?atg?agg?cta 144

Arg?Arg?Lys?Thr?Leu?Gly?Leu?Gly?Arg?His?Cys?Glu?Thr?Met?Arg?Leu

35 40 45

ctt?ttg?cca?gac?tgc?gta?gag?gac?gtg?gtg?aac?ccc?atc?gtc?ctg?cac 192

Leu?Leu?Pro?Asp?Cys?Val?Glu?Asp?Val?Val?Asn?Pro?Ile?Val?Leu?His

50 55 60

ctc?aac?ttc?tcc?ctg?gag?gga?cag?cca?atc?ctc?tca?tcc?cag?aat?ctg 240

Leu?Asn?Phe?Ser?Leu?Glu?Gly?Gln?Pro?Ile?Leu?Ser?Ser?Gln?Asn?Leu

65 70 75 80

cgc?cct?gtg?ctg?gcc?acg?ggc?tcg?cag?gac?cac?ttc?att?gcc?tcc?ctc 288

Arg?Pro?Val?Leu?Ala?Thr?Gly?Ser?Gln?Asp?His?Phe?Ile?Ala?Ser?Leu

85 90 95

ccc?ttt?gag?aag?aac?tgc?gga?caa?gat?cgc?ctg?tgt?gag?ggg?gac?ctg 336

Pro?Phe?Glu?Lys?Asn?Cys?Gly?Gln?Asp?Arg?Leu?Cys?Glu?Gly?Asp?Leu

100 105 110

agc?atc?agc?ttc?aac?ttc?tcg?ggc?ttg?aat?acc?ctg?ctg?gtg?ggg?ctc 384

Ser?Ile?Ser?Phe?Asn?Phe?Ser?Gly?Leu?Asn?Thr?Leu?Leu?Val?Gly?Leu

115 120 125

tcc?ctg?gag?ctc?aca?gtg?aca?gtg?acc?gtg?cgg?aat?gag?ggc?gag?gac 432

Ser?Leu?Glu?Leu?Thr?Val?Thr?Val?Thr?Val?Arg?Asn?Glu?Gly?Glu?Asp

130 135 140

tcc?tat?ggg?acc?gcc?atc?acc?ctc?tac?tac?cca?gca?ggg?cta?tcc?tac 480

Ser?Tyr?Gly?Thr?Ala?Ile?Thr?Leu?Tyr?Tyr?Pro?Ala?Gly?Leu?Ser?Tyr

145 150 155 160

agg?cgg?gtg?tcg?ggc?cag?aca?caa?ccc?tgg?cag?cgc?ccc?ctg?cac?ctc 528

Arg?Arg?Val?Ser?Gly?Gln?Thr?Gln?Pro?Trp?Gln?Arg?Pro?Leu?His?Leu

165 170 175

gca?tgt?gag?gct?gta?cct?acc?gag?agc?gag?ggc?ttg?agg?agt?acc?agc 576

Ala?Cys?Glu?Ala?Val?Pro?Thr?Glu?Ser?Glu?Gly?Leu?Arg?Ser?Thr?Ser

180 185 190

tgc?agc?gtc?aac?cac?ccc?atc?ttc?caa?ggg?ggt?gct?cag?ggc?act?ttc 624

Cys?Ser?Val?Asn?His?Pro?Ile?Phe?Gln?Gly?Gly?Ala?Gln?Gly?Thr?Phe

195 200 205

gta?gtc?aag?ttc?gat?gtc?tcc?tcc?aag?gcc?agc?ctg?ggt?gac?agg?ttg 672

Val?Val?Lys?Phe?Asp?Val?Ser?Ser?Lys?Ala?Ser?Leu?Gly?Asp?Arg?Leu

210 215 220

ctc?atg?ggg?gcc?agt?gcc?agc?agt?gag?aat?aat?aag?cct?gcg?agc?aac 720

Leu?Met?Gly?Ala?Ser?Ala?Ser?Ser?Glu?Asn?Asn?Lys?Pro?Ala?Ser?Asn

225 230 235 240

aag?acc?tcc?ttt?gag?ctg?gaa?ctg?cca?gtg?aaa?tac?gct?gtc?tac?atg 768

Lys?Thr?Ser?Phe?Glu?Leu?Glu?Leu?Pro?Val?Lys?Tyr?Ala?Val?Tyr?Met

245 250 255

atg?atc?aca?agg?cac?gaa?ggc?tcc?acc?agg?ttc?ttc?aac?ttt?tcc?act 816

Met?Ile?Thr?Arg?His?Glu?Gly?Ser?Thr?Arg?Phe?Phe?Asn?Phe?Ser?Thr

260 265 270

tcc?gct?gag?aag?agc?agc?aaa?gag?gcc?gag?cac?cgc?tat?cgg?gtg?aac 864

Ser?Ala?Glu?Lys?Ser?Ser?Lys?Glu?Ala?Glu?His?Arg?Tyr?Arg?Val?Asn

275 280 285

aac?ctg?agt?ctg?cga?gat?gtg?gcc?gtc?agc?gtg?gac?ttc?tgg?gcc?ccc 912

Asn?Leu?Ser?Leu?Arg?Asp?Val?Ala?Val?Ser?Val?Asp?Phe?Trp?Ala?Pro

290 295 300

gtg?cag?ctg?aac?gga?gca?gct?gtg?tgg?gac?gtg?gcg?gtg?gag?gcc?cct 960

Val?Gln?Leu?Asn?Gly?Ala?Ala?Val?Trp?Asp?Val?Ala?Val?Glu?Ala?Pro

305 310 315 320

gcc?cag?agc?ctg?ccc?tgt?gcg?cgg?gag?agg?gaa?cct?ccg?agg?acc?tct 1008

Ala?Gln?Ser?Leu?Pro?Cys?Ala?Arg?Glu?Arg?Glu?Pro?Pro?Arg?Thr?Ser

325 330 335

gac?ctg?agc?cgg?gtc?ccg?ggg?agt?ccc?gtg?ctg?gac?tgc?agc?gtt?gcg 1056

Asp?Leu?Ser?Arg?Val?Pro?Gly?Ser?Pro?Val?Leu?Asp?Cys?Ser?Val?Ala

340 345 350

cac?tgc?ctg?agg?ttc?cgc?tgc?cac?atc?ccc?tcc?ttc?agc?gcc?aag?gag 1104

His?Cys?Leu?Arg?Phe?Arg?Cys?His?Ile?Pro?Ser?Phe?Ser?Ala?Lys?Glu

355 360 365

gag?ctc?cac?ttc?acc?ctg?aag?ggc?aac?ctc?agc?ttc?gcc?tgg?gtc?agc 1152

Glu?Leu?His?Phe?Thr?Leu?Lys?Gly?Asn?Leu?Ser?Phe?Ala?Trp?Val?Ser

370 375 380

cag?atg?ctg?caa?aag?aag?gtg?tcg?gtg?gtg?agt?gtg?gcc?gag?atc?acc 1200

Gln?Met?Leu?Gln?Lys?Lys?Val?Ser?Val?Val?Ser?Val?Ala?Glu?Ile?Thr

385 390 395 400

ttc?aac?agg?gcc?gtg?tac?tcc?caa?gtt?ccg?ggc?gag?gag?ccc?ttt?atg 1248

Phe?Asn?Arg?Ala?Val?Tyr?Ser?Gln?Val?Pro?Gly?Glu?Glu?Pro?Phe?Met

405 410 415

aga?gcc?cag?gtg?gag?acg?gtg?ctg?gag?gag?tat?gag?gag?cac?gac?ccc 1296

Arg?Ala?Gln?Val?Glu?Thr?Val?Leu?Glu?Glu?Tyr?Glu?Glu?His?Asp?Pro

420 425 430

gtc?ccc?ctg?gtg?gtg?ggc?agc?tgt?gtg?ggc?ggc?ctg?ctg?ctg?ctg?gct 1344

Val?Pro?Leu?Val?Val?Gly?Ser?Cys?Val?Gly?Gly?Leu?Leu?Leu?Leu?Ala

435 440 445

ctc?atc?tca?gcc?acc?ctg?tac?aag?ctt?ggc?ttc?ttc?aag?cgc?cgg?tac 1392

Leu?Ile?Ser?Ala?Thr?Leu?Tyr?Lys?Leu?Gly?Phe?Phe?Lys?Arg?Arg?Tyr

450 455 460

aag?gag?atg?ctg?ggc?gag?aaa?ccg?gga?gac?gcg?gcc?acc?ttc?ccc?ggg 1440

Lys?Glu?Met?Leu?Gly?Glu?Lys?Pro?Gly?Asp?Ala?Ala?Thr?Phe?Pro?Gly

465 470 475 480

gag?gac?gcc?agc?tgc?ggg?gct?tca?gat?ttg?cct?ttg?tcc?cag?tg 1484

Glu?Asp?Ala?Ser?Cys?Gly?Ala?Ser?Asp?Leu?Pro?Leu?Ser?Gln

485 490

<210>103

<211>494

<212>PRT

＜213〉rabbit

<400>103

Asp?Val?Gln?Ser?Ser?Ile?Ser?Tyr?Asp?Leu?Ala?Leu?Asp?Pro?Gly?Arg

1 5 10 15

Leu?Val?Ser?Arg?Ala?Ile?Phe?Gln?Glu?Thr?Gln?Asn?Gln?Thr?Leu?Thr

20 25 30

Arg?Arg?Lys?Thr?Leu?Gly?Leu?Gly?Arg?His?Cys?Glu?Thr?Met?Arg?Leu

35 40 45

Leu?Leu?Pro?Asp?Cys?Val?Glu?Asp?Val?Val?Asn?Pro?Ile?Val?Leu?His

50 55 60

Leu?Asn?Phe?Ser?Leu?Glu?Gly?Gln?Pro?Ile?Leu?Ser?Ser?Gln?Asn?Leu

65 70 75 80

Arg?Pro?Val?Leu?Ala?Thr?Gly?Ser?Gln?Asp?His?Phe?Ile?Ala?Ser?Leu

85 90 95

Pro?Phe?Glu?Lys?Asn?Cys?Gly?Gln?Asp?Arg?Leu?Cys?Glu?Gly?Asp?Leu

100 105 110

Ser?Ile?Ser?Phe?Asn?Phe?Ser?Gly?Leu?Asn?Thr?Leu?Leu?Val?Gly?Leu

115 120 125

Ser?Leu?Glu?Leu?Thr?Val?Thr?Val?Thr?Val?Arg?Asn?Glu?Gly?Glu?Asp

130 135 140

Ser?Tyr?Gly?Thr?Ala?Ile?Thr?Leu?Tyr?Tyr?Pro?Ala?Gly?Leu?Ser?Tyr

145 150 155 160

Arg?Arg?Val?Ser?Gly?Gln?Thr?Gln?Pro?Trp?Gln?Arg?Pro?Leu?His?Leu

165 170 175

Ala?Cys?Glu?Ala?Val?Pro?Thr?Glu?Ser?Glu?Gly?Leu?Arg?Ser?Thr?Ser

180 185 190

Cys?Ser?Val?Asn?His?Pro?Ile?Phe?Gln?Gly?Gly?Ala?Gln?Gly?Thr?Phe

195 200 205

Val?Val?Lys?Phe?Asp?Val?Ser?Ser?Lys?Ala?Ser?Leu?Gly?Asp?Arg?Leu

210 215 220

Leu?Met?Gly?Ala?Ser?Ala?Ser?Ser?Glu?Asn?Asn?Lys?Pro?Ala?Ser?Asn

225 230 235 240

Lys?Thr?Ser?Phe?Glu?Leu?Glu?Leu?Pro?Val?Lys?Tyr?Ala?Val?Tyr?Met

245 250 255

Met?Ile?Thr?Arg?His?Glu?Gly?Ser?Thr?Arg?Phe?Phe?Asn?Phe?Ser?Thr

260 265 270

Ser?Ala?Glu?Lys?Ser?Ser?Lys?Glu?Ala?Glu?His?Arg?Tyr?Arg?Val?Asn

275 280 285

Asn?Leu?Ser?Leu?Arg?Asp?Val?Ala?Val?Ser?Val?Asp?Phe?Trp?Ala?Pro

290 295 300

Val?Gln?Leu?Asn?Gly?Ala?Ala?Val?Trp?Asp?Val?Ala?Val?Glu?Ala?Pro

305 310 315 320

Ala?Gln?Ser?Leu?Pro?Cys?Ala?Arg?Glu?Arg?Glu?Pro?Pro?Arg?Thr?Ser

325 330 335

Asp?Leu?Ser?Arg?Val?Pro?Gly?Ser?Pro?Val?Leu?Asp?Cys?Ser?Val?Ala

340 345 350

His?Cys?Leu?Arg?Phe?Arg?Cys?His?Ile?Pro?Ser?Phe?Ser?Ala?Lys?Glu

355 360 365

Glu?Leu?His?Phe?Thr?Leu?Lys?Gly?Asn?Leu?Ser?Phe?Ala?Trp?Val?Ser

370 375 380

Gln?Met?Leu?Gln?Lys?Lys?Val?Ser?Val?Val?Ser?Val?Ala?Glu?Ile?Thr

385 390 395 400

Phe?Asn?Arg?Ala?Val?Tyr?Ser?Gln?Val?Pro?Gly?Glu?Glu?Pro?Phe?Met

405 410 415

Arg?Ala?Gln?Val?Glu?Thr?Val?Leu?Glu?Glu?Tyr?Glu?Glu?His?Asp?Pro

420 425 430

Val?Pro?Leu?Val?Val?Gly?Ser?Cys?Val?Gly?Gly?Leu?Leu?Leu?Leu?Ala

435 440 445

Leu?Ile?Ser?Ala?Thr?Leu?Tyr?Lys?Leu?Gly?Phe?Phe?Lys?Arg?Arg?Tyr

450 455 460

Lys?Glu?Met?Leu?Gly?Glu?Lys?Pro?Gly?Asp?Ala?Ala?Thr?Phe?Pro?Gly

465 470 475 480

Glu?Asp?Ala?Ser?Cys?Gly?Ala?Ser?Asp?Leu?Pro?Leu?Ser?Gln

485 490

<210>104

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>104

tgtccaggac?aagagatgga?cattgc 26

<210>105

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>105

gagctatttc?atagcaagaa?tggg 24

<210>106

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>106

tatagcatag?cgaatgatcc 20

<210>107

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>107

atggtccgtg?gagttgtgat?c 21

<210>108

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: primer

<400>108

tcgagatcca?ccaaactgca?c 21

<210>109

<211>14

<212>PRT

＜213〉monkey

<400>109

Asn?Leu?Asp?Val?Glu?Glu?Pro?Thr?Ile?Phe?Gln?Glu?Asp?Ala

1 5 10

<210>110

<211>14

<212>PRT

＜213〉monkey

<400>110

Asn?Leu?Asp?Val?Glu?Glu?Pro?Thr?Ile?Phe?Xaa?Glu?Asp?Ala

1 5 10

<210>111

<211>15

<212>PRT

＜213〉monkey

<400>111

Phe?Asn?Leu?Asp?Val?Glu?Glu?Pro?Thr?Ile?Phe?Gln?Glu?Asp?Ala

1 5 10 15

<210>112

<211>17

<212>PRT

＜213〉homo sapiens

<400>112

Phe?Asn?Leu?Asp?Val?Glu?Glu?Pro?Thr?Ile?Phe?Gln?Glu?Asp?Ala?Gly

1 5 10 15

Gly

<210>113

<211>16

<212>PRT

＜213〉homo sapiens

<400>113

Phe?Asn?Leu?Asp?Thr?Glu?Glu?Leu?Thr?Ala?Phe?Val?Asp?Ser?Ala?Gly

1 5 10 15

<210>114

<211>17

<212>PRT

＜213〉homo sapiens

<400>114

Phe?Asn?Leu?Asp?Thr?Glu?Asn?Ala?Met?Thr?Phe?Gln?Glu?Asn?Ala?Arg

1 5 10 15

Gly

Claims

1. method that promotes the exercise recovery after the spinal cord injury comprises the anti-α of the victim's effective dose that gives spinal cord injury _dThe step of monoclonal antibody.

2. the process of claim 1 wherein that part is selected from ICAM-R and VCAM-1.

3. method that suppresses the motion infringement after the spinal cord injury comprises the anti-α of the victim's effective dose that gives spinal cord injury _dThe step of monoclonal antibody.

4. the method for claim 3, wherein part is selected from ICAM-R and VCAM-1.

5. dyskinetic method that limits after the spinal cord injury comprises the anti-α of the victim's effective dose that gives spinal cord injury _dThe step of monoclonal antibody.

6. the method for claim 5, wherein part is selected from ICAM-R and VCAM-1.

7. one kind is limited the autonomic nerve after the spinal cord injury and the method for sensory disturbance, comprises the anti-α of the victim's effective dose that gives spinal cord injury _dThe step of monoclonal antibody.

8. the method for claim 7, wherein part is selected from ICAM-R and VCAM-1.

9. any one method among the claim 1-8, wherein anti-α _dMonoclonal antibody is by the hybridoma secretion that is selected from 217L or 226H.

10. any one method among the claim 1-8, wherein anti-α _dMonoclonal antibody combines α with 217L or 226H competition _d

11. any one method among the claim 1-8, wherein anti-α _dMonoclonal antibody suppresses α _dWith α _dThe part combination.

12. any one method among the claim 1-8, wherein spinal cord injury comprises the spinal cord compression.